Title: Purinergic control of neuroinflammation and neuroprotection by the blockade of P2 receptors under excitotoxic conditions in the hippocampus
Authors: Simões, Ana Patrícia Figueiredo Rocha 
Orientador: Cunha, Rodrigo A.
Keywords: Receptores purinérgicos;Receptores P2;Adonosina
Issue Date: 27-Apr-2012
Citation: SIMÕES, Ana Patrícia Figueiredo Rocha - Purinergic control of neuroinflammation and neuroprotection by the blockade of P2 receptors under excitotoxic conditions in the hippocampus [em linha]. Coimbra : [s.n.], 2012. Disponível na WWW:<http://hdl.handle.net/10316/19898>. Tese de doutoramento
Abstract: Purine receptors are involved in the pathophysiology of most disorders of the central nervous system (CNS) taking part in the early synaptic dysfunction and being in the genesis and propagation of the inflammatory response in the brain. However, the different ways that ATP and adenosine receptors contribute to the evolution or to the arrest of deleterious conditions are still being unraveled. The broad spectrum of neuroprotection afforded with the blockade of adenosine A2A receptors under different adverse conditions in the brain can be underlain by a general mechanism through which these receptors may operate. One possibility is the control of neuroinflammation, a common event in most CNS disorders. However, while in the peripheral nervous system A2A receptors have a well established role as a “stop signal” of the inflammatory cascade, it is not clear whether these receptors trigger or arrest the reactivity of microglia, the brain immune-competent cells. To tackle this question, it was tested if the blockade of A2A receptors could prevent biochemical and morphological consequences of neuroinflammation triggered by the systemic administration of lipopolysaccharide (LPS). In this work it is shown that the intracerebroventricular injection of a selective A2A receptor antagonist (SCH58261) was able to prevent the LPS-induced recruitment of activated microglial cells and the release of the pro-inflammatory cytokine interleukin-1β in the hippocampus. Moreover, SCH58261 also prevented the LPS-induced activation of mitogenactivated protein kinases (MAPKs) such as c-Jun N-terminal kinases (JNK) and p38 and the activation of caspase-3, a key mediator of apoptosis. These results indicate a tight control mainly of the genesis of neuroinflammation by the blockade of A2A receptors. Therefore, it was next investigated if A2A receptors were also able to control the direct effects on neurons of the pro-inflammatory cytokines IL-1β and tumor necrosis factor-α (TNF-α), known to be important effectors of neuroinflammation-induced deleterious consequences in the hippocampus. To answer this question, cultured hippocampal neurons were exposed for different periods to different concentrations of these cytokines and their activation of MAPKs was evaluated by Western blot and immunocytochemistry. Both TNF-α and IL-1β increased the phosphorylation (i.e. the activation) of p38 in neurons and IL-1β also increased the phosphorylation of JNK. In addition, the exposure of hippocampal neurons to IL-1β just before adding glutamate increased the susceptibility of cells to glutamate-mediated excitotoxicity. The blockade of A2A receptors with SCH58261 abrogated the activation of
MAPKs induced by IL-1β and prevented the IL-1β-induced exacerbation of excitotoxicity. Taking advantage of single cell calcium imaging it was found that IL-1β increased both the calcium entry and the calcium deregulation caused by glutamate exposure in hippocampal neurons. Pre-incubation of cells with SCH58261 also prevented this effect of IL-1β. Thus, it is concluded that the antagonism of A2A receptors can control not only the genesis of neuroinflammation in vivo but also the direct effects of pro-inflammatory cytokines on neurons, which gives a further insight into the mechanisms operated by these receptors under pathological conditions. In addition to adenosine receptors, the ATP (P2) receptors are involved in the pathophysiology of brain cells in several brain disorders, when the extracellular levels of ATP are significantly raised. In particular, the pharmacological blockade or the genetic deletion of P2Y1 receptors conferred a robust neuroprotection against the toxicity induced by the Aβ1-42 peptide (an Alzheimer’s disease related peptide) in hippocampal neuronal cultures and against Aβ1-42-induced early loss of synaptic markers and mnemonic deficits in rodents. Moreover, the antagonism of P2Y1 receptors ameliorates the consequences of ischaemic/hypoxic insults to hippocampal slices. Given the involvement of neuroinflammation in the deleterious effects of the above conditions, it was hypothesized that the blockade of these receptors could not only control synaptotoxicity but also neuroinflammation. To begin answering this question it was investigated whether blocking the P2Y1 receptors could prevent the direct effects of proinflammatory cytokines on hippocampal neuronal cultures. The results showed that the selective antagonism of P2Y1 receptors either prevented or attenuated the IL-1β-mediated effects in hippocampal neurons but, importantly, it prevented the effects of glutamate per se. Thus it was investigated if the same occurred upon different excitotoxic stimuli to hippocampal neurons. The results obtained show that neurons are protected against the toxicity induced by N-methyl-D-aspartic acid (NMDA) or by quinolinic acid when exposed in the presence of a general P2 receptor antagonist or in the presence of a selective P2Y1 receptor antagonist. This suggests a coupling between P2Y1 receptors and the neurodegeneration mediated by glutamate NMDA receptors. An impaired function of glutamate receptors is implicated in the pathophysiology of chronic neurodegenerative diseases such as epilepsy and in the excitotoxic environment of ischaemia. The previous results obtained with the antagonism of P2Y1 receptors prompted the testing of the blockade of these receptors in animal models of temporal lobe epilepsy and stroke. Epileptic seizures were induced in rats by the intraperitoneal administration of kainate, which results in a clear neurodegeneration of hippocampal neurons. In this work it is shown that kainate-induced seizures cause a long-term modification of the density of most P2 receptors, in both synaptic and glial membranes, predicting a central role of these receptors in the aberrant neurotransmission observed in the brain of epileptic animals. In this model, the blockade of P2Y1 receptors through the intracerebralventricular administration of a selective antagonist (MRS2500) clearly prevented the early kainate-induced activation of microglia and the loss of synaptic markers in the hippocampus. Moreover, it also attenuated the seizureinduced neurodegeneration in hippocampal circuits. Likewise, in a mice model of focal ischaemia generated by the permanent occlusion of the middle cerebral artery, the general blockade of P2 receptors or the selective antagonism of P2Y1 receptors prevented both the ischaemia-induced lesion in the brain and the resultant mnemonic deficits. Overall, the work presented in this thesis provides evidence supporting the control of neuroinflammation by the blockade of A2A and of P2Y1 receptors and also the control of excitotoxicity by the antagonism of P2Y1 receptors in the hippocampus, contributing to the unraveling of the mechanisms through which these receptors may operate to aggravate deleterious conditions in the brain
Description: Tese de doutoramento em Ciências da Saúde, no ramo de Ciências Biomédicas, apresentada à Faculdade de Farmácia da Universidade de Coimbra
URI: http://hdl.handle.net/10316/19898
Rights: openAccess
Appears in Collections:FFUC- Teses de Doutoramento

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