Thèses sur le sujet « Adenosine receptors, A1, A2B »

Adenosine is a nucleoside that plays an important role in the regulation of contractility in the heart. Adenosine receptors are G-protein coupled and those implicated in regulation of contractility are presumed to act via modulating the activity of adenylyl cyclase and cAMP content of cardiomyocytes. Adenosine A1 receptors (A1R) reduce the contractile response of the myocardium to β-adrenergic stimulation. This is known as anti adrenergic action. The A2A adenosine receptor (A2AR) has the opposite effect of increasing contractile responsiveness of the myocardium. The A2AR also appears to attenuate the effects of A1R. The effects of these receptors have been primarily studied in the rat heart and with the utilization of cardiomyocyte preparations. With the increasing use of receptor knockout murine models and murine models of various pathological states, it is of importance to comprehensively study the effects of adenosine receptors on regulation of contractility in the murine heart. The following studies examine the adenosinergic regulation of myocardial contractility in isolated murine hearts. In addition, adenosinergic control of contractility is examined in hearts isolated from A2AR knockout animals. Responses to adenosinergic stimulation in murine isolated hearts are found to be comparable to those observed in the rat, with A1R exhibiting an anti adrenergic action and A2AR conversely enhancing contractility. A significant part of the A2AR effect was found to occur via inhibition of the A1R antiadrenergic action. A part of the anti adrenergic action of A1R has previously been shown to be the result of protein phosphatase 2A activation and localization to membranes. Additional experiments in the present study examine the effect of adenosinergic signaling on PP2A in myocardial extracts from wild type and A2AR knockout hearts. A2AR activation was found to decrease the activity of PP2A and enhance localization of the active enzyme to the cytosol; away from its presumed sites of action. In the A2AR knockout the response to A1R activation was enhanced compared with the wild type and basal PP2A activity was reduced. It is concluded that A2AR modulation of PP2A activity may account for the attenuation of the A1R effect by A2AR observed in the contractile studies.

A cafeína é o psicoestimulante mais consumido mundialmente. Sua ação farmacológica consiste em bloquear os receptores de adenosina A1 e A2A. A administração crônica de cafeína previne déficits cognitivos decorrentes da idade e em modelos experimentais da doença de Alzheimer. Esses efeitos preventivos são também observados pela administração do antagonista seletivo do receptor de adenosina A2A. Nesse trabalho investigou-se a participação dos receptores de adenosina A1 e A2A na prevenção dos déficits cognitivos induzidos por escopolamina. Também foi investigado se a manipulação dos receptores A2A teria algum impacto na memória em camundongos naive. Para isso, foram utilizadas três tarefas comportamentais que avaliaram três tipos de memória: a tarefa de reconhecimento de objetos (RO), a esquiva inibitória (EI) e o labirinto em Y. A administração intraperitoneal de escopolamina (1,0 mg/kg) prejudicou o desempenho da memória de curto prazo nas três tarefas utilizadas. O antagonista de receptores A1 (DPCPX, 1,0 mg / kg, i.p.) preveniu a amnésia induzida por escopolamina no RO e na EI, enquanto o antagonista de receptores A2A (SCH 58261, 0,5 mg / kg, i.p.) preveniu a amnésia em todos os testes. Além disso, ambos os antagonistas não apresentaram efeitos sobre a memória ou a locomoção em animais naive. Também foi observado que a ativação dos receptores A2A, a partir da administração de CGS 21680 (0,1 mg/kg, i.p.) antes da sessão de treino, foi suficiente para provocar prejuízos na memória em animais naive também nas três tarefas, e este efeito foi prevenido por meio da administração de SCH 58261 (0,5 mg/kg, i.p.). Por fim, a administração intracerebroventricular (i.c.v) de CGS 21680 (50 nmol) também prejudicou o desempenho dos animais na tarefa de RO. Em conjunto, estes resultados sugerem que a ativação dos receptores A2A é suficiente para provocar déficits de memória e ainda sugerem que os receptores A1 também participam de maneira seletiva no controle dos déficits de memória relacionados ao sistema colinérgico.
Caffeine, a non-selective adenosine receptor antagonist, prevents memory deficits, an effect mimicked by adenosine A2A receptor (A2AR), but not receptor A1 (A1R), antagonists upon aging and Alzheimer´s disease. We tested if A2AR were also necessary for the memory impairment upon direct perturbation of the cholinergic system with scopolamine and if A2AR activation was sufficient to trigger memory deficits in naive mice using 3 tests, to probe for short-term memory, namely the object recognition task, inhibitory avoidance and modified Y-maze. The intra-peritoneal (i.p.) administration of scopolamine (1.0 mg/kg) impaired short-term memory performance in 3 tests, namely the object recognition task, inhibitory avoidance and modified Y-maze. The scopolamine-induced amnesia was prevented by the A2AR (SCH 58261, 0.5 mg/kg, i.p.) as well as by A1R antagonist (DPCPX, 1 mg/kg, i.p.) in all tests, except for the modified Y-maze, and both antagonists were devoid of effects on memory or locomotion in naive rats. Notably, the activation of A2AR with CGS 21680 (0.1 mg/kg, i.p.) before the training session was sufficient to trigger memory impairment in the 3 tests in naive mice, and effect prevented by SCH 58261 (0.5 mg/kg, i.p.). Furthermore, the intracerebroventricular administration of CGS 21680 (50 nmol) also impaired recognition memory in the object recognition task. These results show that A2AR are necessary and sufficient to trigger memory impairment and they further suggest that A1R might also be selectively engaged to control the cholinergic driven memory impairment.

2016 - 2017
The adenosinergic pathway plays a critical role in cancer development and progression, as well as in drug resistance to chemotherapy and/or targeted-therapy. The goal of this PhD thesis was to investigate and fully characterize the role of CD73/adenosine A2A-A2B receptors axis in cancer, highlighting the therapeutic potential of inhibitors of the adenosinergic pathway. We firstly characterized the mechanism/s by which A2BR promotes immunosuppression and angiogenesis in tumor-bearing hosts, focusing on the role of myeloid-derived suppressor cells (MDSCs) and cancer-associated fibroblasts (CAFs). The results revealed that treatment of melanoma-bearing mice with Bay60-6583, a selective A2BR agonist, is associated with 1. increased tumor VEGF-A expression and vessel density, and 2. increased accumulation of tumor-infiltrating CD11b+Gr1+cells (MDSCs). MDSCs strongly contribute to the immunosuppressive and angiogenic effects of Bay60-6583. Melanoma-bearing mice treated with a selective A2BR antagonist PSB1115 showed reduced tumor growth compared to controls and this effect was associated with reduced tumor angiogenesis, low levels of MDSCs and increased number of tumor-infiltrating CD8+ T cells. Furthermore, blockade of A2BR increased the anti-tumor effects of VEGF-A inhibitors. Next, we verified that A2BR activation also drives fibroblasts activation within melanoma tissues, by increasing the number of FAP positive cells within tumor lesions. FAP is a common marker of activated fibroblasts also named cancer-associated fibroblasts. These cells produce and secrete various tumor-promoting factors, including fibroblast growth factor (FGF)-2 and CXCL12 or stromal-derived factor 1 α (SDF1α), that were increased both in melanoma tissue and fibroblasts isolated from melanoma tissue or from skin upon Bay60-6583 treatment. Bay60-6583-induced FGF-2 from fibroblasts contributed to melanoma cells proliferation. The CXCL12/CXCR4 pathway, instead, was involved in the pro-angiogenic effects of A2BR agonist, but not in its immunosuppressive effects. These effects were significantly blocked by the A2BR antagonists PSB1115. Taken together, these data elucidate the pivotal role of A2BR in establishing a positive cross-talk between tumor-infiltrating immune cells, fibroblasts and endothelial cells that sustain tumor growth, reinforcing the therapeutic potential of A2BR blockers for cancer therapy. ... [edited by Author]
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Adenosine receptors are members of the G protein coupled receptor (GPCR) superfamily. Neurochemical and electrophysiological experiments point to a neuromodulatory role for adenosine. Adenosine inhibits the release of several neurotransmitters in brain by pre- and postsynaptic actions at adenosine A1 receptors. In this thesis the pharmacology and modulation of adenosine A1 receptors in the central nervous system (CNS) of different species was characterised. Rat renal adenosine A1 receptors were also studied. In addition the central penetration by adenosine A1 receptor antagonists following peripheral administration was investigated. An in vitro [3H]DPCPX ligand binding assay utilising a range of receptor agonists and antagonists, were used to compare the pharmacology of rat, mouse, guinea pig and human adenosine A1 receptors. Standard xanthine-based adenosine antagonists had 10-fold lower affinity in human and guinea pig in comparison with rat and mouse. Several novel nonxanthine A1 antagonists from the Fujisawa Pharmaceutical Co. Ltd., retained high affinity for adenosine A1 receptors in all species, including human. Modulation of ligand binding to adenosine A1 and A2a receptors by Gpp(NH)p and magnesium was examined. In common with other GPCRs, agonists bind to high and low affinity states, with the equilibrium modified by GTP analogues and magnesium, whereas effects on antagonist affinity are minimal. Agonist radioligands under the conditions used labelled predominantly the high affinity state of adenosine A1 and A2a receptors. Gpp(NH)p and magnesium have essentially inverse effects on radioligand (agonists and antagonists) binding to both adenosine A1 and A2a receptors. In addition their effects on [3H]agonist and [3H]antagonist binding are generally opposite, which is consistent with effects observed for other GPCRs. To act centrally, compounds must cross the blood brain barrier (BBB). A modified radioreceptor assay, involving a denaturation step to overcome the lipophilic nature of adenosine A1 receptor antagonists was developed. This assay accurately measures the central penetration of adenosine A1 receptor antagonists. Adenosine A1 antagonists administered peripherally at equipotent behavioural doses, were found to be present in brain, at concentrations between 50-500 fold greater than in vitro Ki values for adenosine A1 receptors. In addition, there was an excellent association between antagonist affinity in vitro and antagonist brain concentrations at the equipotent behavioural dose. Adenosine and its receptors are widely distributed throughout the body. It is important to determine if peripheral adenosine A1 receptors are identical to those in the CNS. Renal adenosine A1 receptors were examined using radioligand binding, in vitro autoradiography and in situ hybridisation. Even using this combined approach, identification of adenosine A1 receptors in rat kidney was not possible. Alzheimer's disease involves a myriad of neurochemical changes and neurotransmitter deficits. By antagonising the actions of endogenous adenosine, A1 receptor antagonists could compensate for some of these deficits, by enhancing synaptic facilitation and increasing neurotransmitter release. Analogues of FK453, a non-xanthine adenosine A1 antagonist, which are BBB permeable and potent and selective for human adenosine A1 receptors, could prove to be clinically useful compounds, by enhancing cognition, in conditions such as Alzheimer's disease.

Protecting hearts from damage sustained during myocardial ischaemia and reperfusion remains an ongoing challenge. Despite successful findings with animal models, the task of trialling and effectively translating experimental findings from laboratory to patients has proven difficult, therefore treatments and clinical therapies are still urgently needed to protect the heart and improve cardiac functional outcome. Previous research has implicated adenosine and opioid receptor participation in the protective response preceding or following myocardial infarction, with evidence of potential cross-talk between receptors. This project aimed to investigate whether A1 adenosine (A1AR) and δ-opioid receptor (δ-OR) dependent cytoprotection and prosurvival kinase activation in murine hearts share common dependencies on „cross-talk‟ between both G-protein coupled receptor (GPCR) sub-types and whether these responses involve a common Matrix Metalloproteinase (MMP) and Epidermal Growth Factor Receptor (EGFR) dependent signalling pathway. This was achieved via four inter-related in vitro studies. Study 1: Healthy mouse hearts were cannulated in a Langendorff mode enabling the coronary circulation to be perfused. Hearts were subjected to 25 minutes of global (zero-flow) ischaemia followed by 45 minutes of aerobic reperfusion. The groups investigated included untreated control hearts, hearts receiving the selective A1AR agonist CCPA (± DPCPX, a selective A1AR antagonist, or BNTX, a selective δ-OR antagonist) and hearts receiving the selective δ-OR agonist BW373U86 (± DPCPX, a selective A1AR antagonist, or BNTX, a selective δ-OR antagonist). Agonists were applied 5 minutes pre-ischaemia and the antagonists were administered 10 minutes prior to agonist treatment. Cardiac functional outcomes were assessed via changes in coronary flow, left ventricular (LV) end diastolic pressure and pressure development, and ±dP/dt (± differentials of pressure change with time – indexing lusitropic and inotropic state). Cell death outcomes were also assessed via lactate dehydrogenase (LDH) release. Treatment with either the selective A1AR agonist CCPA, or the selective δ-OR agonist BW373U86, significantly reduced (p≤0.0001 vs. CTRL) LV end-diastolic pressure following ischaemia/reperfusion. Recovery of LV developed pressure (LVDP) was significantly increased following A1AR activation via CCPA (p≤0.0001 vs. CTRL) or δ-OR activation via BW373U86 (p≤0.001 vs. CTRL). Ventricular contractility (+dP/dt) and relaxation (-dP/dt) were also significantly improved with both CCPA (p≤0.0001 vs. CTRL, p≤0.01 vs. CTRL) and BW373U86 (p≤0.001 vs. CTRL, p≤0.001 vs. CTRL). Treatment with CCPA, but not BW373U86, significantly improved the recovery of coronary flow rate at the termination of reperfusion (p≤0.01 vs. CTRL). LDH release (corresponding to cell death) was significantly reduced by both CCPA and BW373U86 (p<0.05 vs. CTRL, p<0.05 vs. CTRL). A1AR or δ-OR inhibition, via the selective antagonists DPCPX and BNTX respectively (applied alone), did not significantly affect the recovery of functional outcomes or cell death relative to control. These results show that cardioprotection against ischaemic injury is induced with activation of A1ARs and δ-ORs, and that endogenous levels of receptor agonists may not be sufficient to induce this response. Protection with CCPA was abolished via cotreatment with either the selective A1AR antagonist DPCPX or the selective δ-OR antagonist BNTX. Conversely protection with BW373U86 administration was negated by co-treatment with either BNTX or DPCPX. This reveals that A1AR dependent cardioprotection is reliant on the activation of δ-ORs, and δ-OR mediated protection is dependent on A1AR activity, confirming essential cross-talk. Study 2: Perfused hearts from study 1 were snap-frozen in liquid nitrogen following the termination of reperfusion. Hearts were homogenised and fractioned to yield cytosolic proteins. Total and phosphorylated levels of Erk1/2 and Akt were subsequently assessed via western immunoblot. Both A1AR and δ-OR stimulation via CCPA and BW373U86 (respectively) did not significantly influence Erk1/2 phosphorylation. Akt phosphorylation, on the other hand, was increased by both CCPA and BW373U86; although only the latter effect achieved statistical significance (p<0.01 vs. CTRL). The A1AR antagonist DPCPX had minimal effect on Erk1/2 and Akt phosphorylation when applied alone. Alternatively inhibition of the δ-OR via BNTX, applied alone, was found to increase both Akt and Erk1/2 phosphorylation, a response in conflict with the existing literature. Co-treatment with the A1AR antagonist DPCPX or the δ-OR antagonist BNTX did not significantly influence Erk1/2 signalling compared to controls. Alternatively Akt phosphorylation was reduced by ~50% relative to control when hearts were co-treated with DPCPX or BNTX applied in conjunction with either the A1AR agonist CCPA or the δ-OR agonist BW373U86. These results imply that both A1ARs and δ-ORs together are necessary to induce protective Akt signalling during ischaemia/reperfusion with either receptor agonist. Study 3: To assess the roles of EGFRs and MMPs in A1AR and δ-OR responses, agonist studies with CCPA and BW373U86 were repeated with co-treatment with the EGFR antagonist AG1478 or the MMP inhibitor GM6001. Functional and cytoprotective outcomes were assessed in perfused hearts subjected to ischaemiareperfusion. The protective response observed with either A1AR and δ-OR stimulation was negated via co-treatment with either AG1478 or GM6001. A1AR and δ-OR dependent recovery of end diastolic pressure, LV developed pressure, +dP/dt and -dP/dt were all repressed via EGFR or MMP inhibition. Moreover, the cytoprotective response conferred by δ-OR activation was completely abolished via co-treatment with AG1478 or GM6001, providing evidence that adenosinergic and opioidergic protection within the myocardium involves an EGFR and MMP dependent pathway. Study 4: Western blot analysis was used to assess changes in Erk1/2 and Akt expression and phospho-regulation in hearts treated with CCPA or BW373U86 in the presence of AG1478 or GM6001. Due to time constraints, data collected previously in our lab was used in this research; therefore the effect of EGFR and MMP inhibition on A1AR dependent Erk1/2 and Akt signalling was assessed in whole heart rather than cytosolic fractions. In these hearts, administration of CCPA significantly elevated both Erk1/2 and Akt phosphorylation, a response negated via co-treatment with either AG1478 or GM6001 (p≤ 0.05 vs. CCPA). Infusion of the selective δ-OR agonist BW373U86 did not significantly alter Erk/1/2 expression or phosphorylation in cytosolic fractions. Despite this, co-treatment with AG1478 reduced Erk1/2 phosphorylation by ~50% compared to the agonist alone (p ≤ 0.05 vs. BW373U86), suggesting an EGFR dependent mechanism. Surprisingly co-treatment with GM6001 did not significantly influence δ-OR mediated Erk1/2 activity. Akt phosphorylation was increased by more than 60% with BW373U86 (p≤0.05 vs. CTRL) and this response was abolished via treatment with the selective EGFR antagonist AG1478 or the selective MMP inhibitor GM6001. This provides further evidence that adenosinergic and opioidergic protective signalling during ischaemia-reperfusion requires the activity of EGFRs and MMPs. Conclusions: As a whole, the present study confirms an essential interaction between ARs and ORs in the heart, with kinase signalling and tissue protection via either A1ARs or -ORs exhibiting common and essential dependencies on activity of both receptors. The basis of this intriguing response remains unclear, although we show that both receptors engage distal kinases (and cardioprotection) in an MMP/EGFR dependent manner, adding an additional level to this novel cross-talk. This research provides further insight into cardioprotective receptor interactions in the heart, potentially leading to the development of new pharmacotherapeutics and improved outcomes for cardiovascular disease patients. Further research is needed to clarify the mechanism behind adenosinergic and opioidergic cross-talk and cardioprotection, potentially exploring membrane signalling, temporal expression of kinases, existence of A1AR/δ-OR dimers/oligomers, and concepts such as dual agonism and potential signalling thresholds for protection.
Thesis (Masters)
Master of Medical Research (MMedRes)
School of Medical Science
Griffith Health
Full Text

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Neurociências, Florianópolis, 2016.
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A guanosina, nucleosídeo derivado da purina guanina, exerce efeitos neuroprotetores e neurotróficos no Sistema Nervoso Central (SNC). Recentemente, um sistema de neurotransmissão purinérgica dos derivados da guanina, ou sistema guanosinérgico, foi proposto. Deste sistema de neurotransmissão, já são conhecidos transportadores de nucleosídeos e enzimas responsáveis pelo metabolismo intra e extracelular, porém os receptores de membrana seletivos para a guanosina ainda não foram claramente caracterizados. Alguns efeitos neuroprotetores da guanosina parecem depender da ativação dos receptores de adenosina A1R e A2AR. Nesta tese, foi avaliada (i) a interação da guanosina com oligômeros de receptores de adenosina A1RA2AR; e (ii) o efeito da guanosina em um modelo animal da doença de Alzheimer. No capítulo I, através da superexpressão heteróloga dos receptores A1 e A2A em células HEK293, demonstramos que a guanosina não altera a colocalização destes receptores na membrana celular e sua heteromerização, quando avaliada por BRET e complementação de fluorescência. A guanosina não tem efeito per se, mas inibe o aumento de Ca2+ intracelular induzido por agonista de A1R (CCPA) em células que expressam A1R. Em células que expressam A1R+A2AR guanosina previne a aumento de Ca2+ induzido por agonista de A1R ou de A2AR. Guanosina induz aumento de AMPc em células A2AR, mas inibe o aumento de AMPc na presença dos agonistas A2AR, adenosina e CGS21680. Experimentos de nanoBRET sugerem que a guanosina desloca a ligação do agonista A2AR (APEC) em células que expressam A2AR e oligômeros A1R+A2AR. O efeito neuroprotetor da guanosina, não é observado em um modelo de isquemia in vitro realizado em fatias hipocampais de camundongos knock-out para o A2AR. Os resultados sugerem que guanosina pode interferir com a sinalização celular ativada por A1R e A2AR, e na presença de agonistas A2AR, guanosina bloqueia a ativação deste receptor. Além disso, o efeito neuroprotetor da guanosina depende da expressão de A2AR. No capítulo II desta tese, o efeito neuroprotetor da guanosina foi avaliado em um modelo de toxicidade do peptídeo beta-amiloide (Aß1-40), um modelo animal da doença de Alzheimer. Guanosina administrada logo após a infusão i.c.v do Aß1-40 e durante 14 dias consecutivos reverteu o déficit cognitivo e o comportamento tipo-anedônico induzidos pelo Aß1- 40. Guanosina preveniu o aumento na captação de glutamato independente de Na+ induzido pelo Aß1-40. A análise dos níveis de purinas mostra que Aß1-40 aumentou os níveis de ADP e ATP no hipocampo dos animais, e que o tratamento com guanosina aumentou os níveis de GDP. Aß1-40 reduziu a expressão de GFAP na região CA1 do hipocampo dos camundongos e guanosina não alterou esse efeito. Não foi observada alteração nos níveis de sinaptofisina no hipocampo dos animais. Os resultados obtidos sugerem que guanosina previne alterações cognitivas e no transporte de glutamato induzidas pelo Aß1-40 em camundongos. Esta tese contribuiu para identificar os sítios extracelulares de interação da guanosina e adicionou evidências sobre o efeito neuroprotetor da guanosina.

Abstract : Guanosine, the guanine-based nucleoside, exerts neurotrophic and neuroprotective effects in the Central Nervous System (CNS). A guaninebased purinergic system - or guanosinergic system ? has been recently proposed. In this neurotransmitter system some components are known, as nucleosides transporters and enzymes responsible for intra and extracellular metabolism. However, a selective extracellular site for guanosine interaction or guanosine receptor has not yet been clearly characterized. Some of guanosine neuroprotective effects seem to be dependent upon the activation of adenosine A1 or A2A receptors. In this study, we investigated (i) the possible guanosine interaction with A1RA2AR oligomers, and (ii) guanosine neuroprotective effect in an animal model of Alzheimer´s disease. Using A1R and A2AR heterologous expression in HEK193 cells, we showed that guanosine did not alter the colocalization of these receptors at the cellular membrane. Guanosine also did not modify A1R-A2AR heteromeric formation, evaluated with BRET and bimolecular fluorescence complementation (BiFC) techniques. Guanosine alone had no effect on intracellular Ca2+ signaling, but it inhibited the increase in Ca2+ induced by an A1R agonist (CCPA) in A1Rcells. In A1R+A2AR-expressing cells, guanosine prevents Ca2+ signaling evoked by A1R or A2AR agonists. Guanosine increased cAMP levels in A2AR cells, but it inhibited cAMP increase induced by adenosine or CGS21680 (A2AR agonists). NanoBRET experiments suggested that guanosine displaced the binding of an A2AR agonist (APEC), both in A2AR and A2AR+A1R cells. Guanosine neuroprotective effect in an in vitro model of ischemia was not observed in hippocampal slices of A2ARknockout mice. Data presented here suggest that guanosine can interfere on A1R and A2AR activated signaling pathways. However, in the presence of an A2AR agonist, guanosine blocks A2AR activation. Furthermore, the neuroprotective effect of guanosine is dependent on A2AR expression. In chapter II, we assessed guanosine neuroprotective effects against amyloid-beta peptide (Aß1-40) intracerebroventricular infusion, an animal model of Alzheimer´s disease. Guanosine treatment soon after Aß1-40 i.c.v infusion and once a day during the 14 consecutive days, inhibited the cognitive deficit and the anhedonic-like behavior induced by Aß1-40. Guanosine prevented the impairment in Na+-independent glutamate transport induced by Aß1-40. Purines levels analysis showed that Aß1-40 increased ATP and ADP at mice hippocampus, and guanosine increased GDP levels. Aß1-40 infusion decreased GFAP at CA1 hippocampal region, an effect not modified by guanosine. No changes in synaptophysin levels were observed. Presented data show that guanosine prevents cognitive alterations and the unbalance in glutamate transmission evoked by Aß1-40 in mice. Therefore, this thesis has contributed to identify extracellular sites to guanosine action and add information on the neuroprotective effects of guanosine.

Existem várias terapias para o controle da dor neuropática, mas os tratamentos atuais têm pouca eficiência e apresentam muitos efeitos colaterais, por isso novas alternativas terapêuticas são necessárias. A Frutose-1, 6-bifosfato (FBP) é um intermediário da via glicolítica que tem várias ações farmacológicas e, recentemente, foi demonstrado o seu efeito anti-hiperalgésico em modelo de dor inflamatória aguda via produção de adenosina. Curiosamente, a adenosina e agonistas do seus receptores A1 e A2A têm efeitos anti-hiperalgésicos em modelos de dor neuropática. Assim, investigamos o efeito anti-hiperalgésico e o mecanismo de ação da FBP em um modelo de dor neuropática induzido por constrição crônica (CCI) do nervo ciático de camundongos Swiss. Sete dias após a cirurgia, os animais foram tratados com diferentes doses de FBP ou adenosina por via oral ou intratecal. Além disso, os tratamentos da FBP e adenosina, por via oral, foram associados a administração de antagonistas dos receptores de adenosina A1 ou A2A, nas vias intraplantar ou intratecal. Por fim, o tratamento por via oral da FBP foi associado a inibidores da via de sinalização do óxido nítrico/GMPcíclico/Proteína quinase G/canais de potássio sensíveis ao ATP (NO/GMPc/PKG /KATP), que já foi demonstrada como via do mecanismo de ação da adenosina. A hiperalgesia mecânica foi avaliada por uma versão eletrônica dos filamentos de von Frey 1, 3, 5 e 7 horas após os tratamentos. A FBP e a adenosina inibiram a hiperalgesia mecânica induzida pela CCI de maneira similar. O mecanismo de ação da FBP parece ser dependente da produção de adenosina, já que seu efeito foi inibido pelos antagonistas dos receptores de adenosina A1 e A2A; além disso, o tratamento com a FBP promove anti-hiperalgesia através da via de sinalização NO/GMPc/PKG/KATP de forma semelhante à adenosina. Assim, demonstrou-se que a FBP tem benefício terapêutico na redução da dor neuropática e que seus mecanismos envolvem a ativação de receptores de adenosina A1 e A2A e a via de sinalização NO/GMPc/PKG/KATP.
There are several therapies for neuropathic pain control, but current treatments are lacking efficacy and have many side effects, so alternatives for this treatment are needed. Fructose-1, 6-bisphosphate (FBP) is an intermediate of the glycolytic pathway that has several pharmacological actions, and more recently it was demonstrated its anti-hyperalgesic effect in acute inflammatory pain model via production of adenosine. Interestingly, adenosine and adenosine A1 and A2A receptor agonists have anti-hyperalgesic effects in neuropathic pain models. Thus, we investigated the anti-hyperalgesic effect and mechanism of action of FBP in a model of chronic constriction injury (CCI) of sciatic nerve-induced neuropathic pain in Swiss mice. Seven days after surgery, animals were treated with FBP or adenosine in oral or intrathecal administration. Moreover, the oral treatments of FBP and adenosine were associated with A1 or A2A adenosine receptor antagonists in intraplantar or intrathecal administration. Finaly, oral treatment of FBP was associated with inhibitors of nitric oxide/cycle GMP/Protein Kinase G/Potassium Channels ATP-sensitive (NO/cGMP/PKG/KATP) signaling pathway, which was already demonstrated as mechanism of action of adenosine. The mechanical hyperalgesia was assessed by an electronic version of von Frey filaments 1, 3, 5 and 7 hours after treatments. FBP and adenosine inhibited the mechanical hyperalgesia induced by CCI in a similar profile. FBP mechanism of action seems to be dependent on adenosine production as its effect was inhibited by adenosine A1 and A2A receptors antagonists, and FBP treatment promote anti-hyperalgesia through NO/cGMP/PKG/KATP signaling pathway similarly to adenosine. Concluding, it was demonstrated that FBP has possible therapeutic application to reduce neuropathic pain, and its mechanisms involve adenosine activation of A1 and A2A receptors and the NO/cGMP/PKG/KATP signaling pathway.

Dans le cerveau, les fonctions de la phosphatase alcaline non spécifique des tissus (TNAP) ne sont pas clairement identifiées. La localisation et l'expression de cette enzyme au niveau neuronal suggère cependant, qu'elle joue un rôle important dans le développement et le fonctionnement du cerveau. Cela est supporté par la présence de graves crises d'épilepsie chez les humains porteurs d'une mutation de la TNAP. Ces crises d'épilepsie sont létales chez les souris KO pour la TNAP. Des études chez la souris montrent que la TNAP pourrait réguler l'inhibition postsynaptique médiée par le GABA et elle pourrait être impliquée dans l'inhibition présynaptique médiée par l'adénosine. L'adénosine est, en partie, synthétisée via la déphosphorylation successive de l'ATP en ADP puis en AMP par des ectonucléotidases. Parmi elles, la TNAP et l'ecto- 5'-nucléotidase (NT5E) catalysent l'hydrolyse de l'AMP en adénosine dans le cortex cérébral. L'adénosine agit principalement au niveau présynaptique par l'intermédiaire des récepteurs A1. Ainsi l'adénosine a une influence sur la transmission synaptique et sur la plasticité synaptique. Ceci pourrait expliquer, en partie, les crises d'épilepsie observées chez les souris KO pour la TNAP. Les deux objectifs principaux de ma thèse ont été : (1) évaluer la contribution de la TNAP dans la production d'adénosine dans le cerveau ; (2) étudier l'influence de l'adénosine sur la plasticité synaptique. Premièrement, l'étude de la contribution de la TNAP dans la production d'adénosine dans le cerveau a été réalisée au moyen de deux approches complémentaires. Une approche métabolomique (spectroscopie RMN du proton) sur des cerveaux entiers de souris KO pour la TNAP a permis de montrer que la TNAP participe, entre autre, à la synthèse d'adénosine dans le cerveau. Une deuxième approche, électrophysiologique sur tranches de cerveaux de souris in vitro, nous permet d'examiner les conséquences de l'inhibition des ectonucléotidases intervenant dans la synthèse de l'adénosine. Elle a révélé que l'inhibition des ectonucléotidases (TNAP et NT5E) ne supprime pas l'effet inhibiteur de l'AMP médiée par les récepteurs A1. Deuxièmement, nous avons étudié l'influence de l'adénosine sur la plasticité synaptique à courte terme. Nous avons enregistré des potentiels de champs dans la couche Ia du cortex piriforme en réponse à des stimulations électriques (3,125 à 100 Hz) présentée avec des fréquences recouvrant la gamme d'oscillations physiologiques. Nos résultats montrent qu'avec de fortes concentrations d'adénosine, la facilitation est accentuée par rapport à celle observée en situation contrôle. Cet effet est observé pour des fréquences supérieures ou égales à 25 Hz. De plus, cette accentuation est d'autant plus grande que la fréquence est élevée (maximum atteint à 100 Hz pour 100 µM). En bloquant l'action de l'adénosine endogène, l'effet contraire est observé : une facilitation déficitaire par rapport au contrôle et dont le défaut est croissant avec la fréquence de stimulation. Tous ces résultats convergent vers l'hypothèse qu'une déficience en TNAP, traduite par une absence d'adénosine, pourrait contribuer au maintien des processus épileptiques générés par un déséquilibre de l'inhibition et de l'excitation dû à une diminution de GABA. L'effet inhibiteur de l'AMP médié par les récepteurs A1 ne serait pas suffisant pour contrecarrer les crises d'épilepsie observées chez les sujets hypophosphatasiques et les souris KO pour la TNAP
The functions of Tissue Nonspecific Alkaline Phosphatase (TNAP) in the brain are not clearly identified. The localization and expression of TNAP at the neuronal level, however, suggests that it plays a prominent role in the development and the function in the brain. This is supported by the presence of severe epileptic seizures in humans carrying TNAP mutation. These epileptic seizures are lethal in TNAP KO mice. Studies in mice show that TNAP could regulate GABA-mediated postsynaptic inhibition and may be involved in presynaptic inhibition mediated by adenosine. Adenosine is, partly, synthesized via the successive dephosphorylation of ATP to ADP and then to AMP by ectonucleotidases. Among them TNAP and ecto-5'-nucleotidase (NT5E) are able to hydrolyze AMP into adenosine. Adenosine acts mainly at the presynaptic level via A1 receptors activation. Adenosine has an influence on synaptic transmission and thus on synaptic plasticity. This could partly explain the epileptic seizures observed in TNAP knock-out mice. The two main purposes of my thesis were: (1) to evaluate the contribution of TNAP in adenosine production in the brain; (2) to study the influence of adenosine on synaptic plasticity. Firstly, the study of the contribution of TNAP in adenosine production in the brain was carried out using two complementary approaches. A metabolomic approach (proton NMR spectroscopy) on whole brains of TNAP KO mice showed that TNAP in involved in adenosine synthesis in the brain. In a second approach, in vitro electrophysiological recordings on mouse brain slices allowed us to examine the consequences of the inhibition of the ectonucleotidases involved in adenosine synthesis. This revealed that inhibition of ectonucleotidases (TNAP and NT5E) did not suppress the inhibitory effect of AMP mediated by A1 receptors. Secondly, we studied the influence of adenosine on short-term synaptic plasticity. Field potentials were recorded in response to electrical stimulations (3.125 to 100 Hz) applied with frequencies encompassing the range of physiological oscillation. Our results show that, with high adenosine concentrations, the facilitation is emphasized compared to that observed in the control situation. This effect is observed for frequencies greater than or equal to 25 Hz. In addition, the higher the frequency, the greater the facilitation. Finally, by blocking the action of endogenous adenosine, the opposite effect was observed: a deficient facilitation with respect to the control, whose defect was increasing with stimulation frequency. All these results converge towards the hypothesis that TNAP deficiency, expressed by absence of adenosine, could contribute to the maintenance of the epileptic processes generated by an imbalance of the neuronal inhibition and the excitation due to a decrease of GABA. AMP inhibitory effect mediated by A1 receptors, would not be sufficient to counteract epileptic seizures observed in hypophosphatasic patients and TNAP KO mice

G protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that perform transmembrane signal transduction. Due to their pivotal role in a wide range of essential physiological functions GPCRs represent a high proportion of all drug targets. High resolution X-ray structures of GPCRs are however underrepresented in the Protein Data Bank. This is due to their instability in detergent, low expression levels and the presence of misfolded receptors in many heterologous expression systems. The objective of this project was to engineer the angiotensin II type 1 receptor (AT1R), a human GPCR, to make it suitable for structural studies. It was determined that detergentsolubilised AT1R was thermostable with antagonist bound with an apparent Tm of ~45°C, which was sufficiently stable for purification without further thermostabilisation by rational mutagenesis. Two expression systems were then evaluated for large-scale production of AT1R, namely baculovirus-mediated expression in insect cells and mammalian expression in HEK293 cells. Radioligand binding assays showed that only the mammalian system produced sufficient quantities of active AT1R for structural studies. Expression in the mammalian system was further optimised to approximately 6 mg/L. An AT1R-GFP fusion was created to examine membrane localisation using confocal laser scanning microscopy, to assay expression levels, to select highly expressing monoclonal cell lines using fluorescence activated flow cytometry and to develop a fluorescence size-exclusion chromatographybased assay to examine the suitability of 12 different ligands for co-crystallization. AT1R was also engineered to facilitate crystallisation, including C-terminal truncations to remove predicted disordered regions and bacteriophage T4-lysozyme being added to the third intracellular loop to provide additional points of contact for crystallisation, which increased the apparent Tm by approximately 10°C. All modified versions of AT1R were assessed for expression, stability and monodispersity. Additionally a rapid western blotting based assay was developed for the detection of unfolded membrane proteins, which will have wide applicability in the field.

The research reported in this Ph.D. thesis, though it has to be deepened, furthers our understanding about the ARs. New AR ligands were developed preferentially belonging to the aminopyridine-3,5-dicarbonitrile series whose structure-activity relationships have been till now poorly investigated. In fact, most of the compounds reported in the literature are included in patent documents. In this work, many derivatives were synthesized and biologically evaluated in binding and functional assays in order to assess affinity and selectivity towards a specific AR subtype, but also to evaluate their pharmacological profile. The data obtained confirmed the peculiarity of this series whose derivatives are not only characterized by a wide range of affinities but interestingly endowed with different degree of efficacies at the different ARs. Based also on molecular modelling studies two set of compounds, named DCP1 and DCP2B were designed preferentially targeting the A1 and A2B AR, respectively. Most of the compounds belonging to the DCP1 set are endowed with high affinity and good selectivity at the hA1 AR. Some selected derivatives showed also an inverse agonist profile with the only exception of compound 15 which emerged as partial agonist. Thus, further modification at the level of the thiazole moiety will be planned in order to evaluate the importance of this substituent for obtaining hA1 AR agonists. Moreover, when evaluated in an oxaliplatin-induced neuropathic pain model in mice, compound 1 (dual A1 inverse agonist/A2A antagonist) resulted to revert allodynia at very low doses after oral administration. This effect is similar to that exerted by both caffeine and other A1/A2A antagonists as well as A1 AR agonists in reverting hyperalgesia and in antinociception. Stability studies in mouse and human plasma on 1 confirmed no significant chemical modification of the compound for the whole duration of the assay. Thus, to clarify this paradoxical data, other molecules belonging to this set, endowed with similar degree of affinities and efficacies with respect to 1, will be tested in this in vivo assay, also to evaluate their potentiality for neuropathic pain treatment. The results obtained on the DCP1 set, especially in the in vivo assay, suggested that the amino-3,5-dicyanopyridine scaffold could be of interest for obtaining non-nucleoside AR agonists as to prevent two serious problems hampering the development of classical adenosine-like agonists: the low oral bioavailability and the short half-life due to the presence of the typical ribose (or ribose-mimicking) moiety. DCP2B set is composed of compounds preferentially targeting the hA2B receptor. Most of them showed to behave as partial agonist, with the best results concerning activity and selectivity of compound 37, which is three fold more potent than the reference agonist BAY60-6583. This result can be considered a real breakthrough since BAY60-6583 is absolutely the sole potent and selective hA2B AR agonist reported in literature. Moreover, it is noteworthy that derivatives 66 and 67, showing high activity at the hA2BAR but scarce selectivity, emerged as the only two amino-3,5-dicyanopyridines reported till now which are endowed with a full agonist profile. With this in mind, a study to clarify the structural requirements which are important in determining the A2B pharmacological profile of these compounds is currently on going. Thus, identification of new selective hA2B agonists could be of help in deepening the medicinal chemistry and the pharmacology related to this still unknown AR subtype. In fact, compound 37 was used as pharmacological tool in an oligodendrocyte precursor cell (OPC) differentiation assay in order to clarify whether the A2B receptor represents a promising target to modulate endogenous remyelinization process in multiple sclerosis patients. Compound 37 inhibits OPC differentiation into oligodendrocytes at lower concentration than those used for BAY60-6583 demonstrating the role of A2B AR in this important physiological process. Moreover, docking studies were performed on selected DCP1 and DCP2B derivatives at A1 and A2B homology receptor models, respectively, showing the best binding poses for compounds 15 and 37 and the most important residues involved in the binding at these two AR subtypes. It seems to clarify the role of R4 and R6 substituents, containing atoms or groups able to engage hydrogen-bonding interactions with the receptors, in modulating affinity and efficacy of the amino-3,5-diyanopyridine derivatives. Moreover, since some affinity was also achieved at the hA2A and hA3 AR with some of the compounds detailed in this thesis, it may be possible that this heterocycle core could be also manipulated to enhance affinity and selectivity for these subtypes. In fact, neither A2A nor A3 agonists belonging to the amino-3,5-dicyanopyridine series has been ever designed. Preliminary studies on the newly synthesized 7-amino-2-phenylpyrazolo[1,5-c]pyrimidine-4-carbonitrile system, derived from molecular complications of the amino-3,5-dicyanopyridine core, were reported. Biological results on a small set of compounds, that bind with good affinity the hA1 AR, indicated that this scaffold suitably decorated could generate new AR ligands.

Dissertação de mestrado em Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.
Major depressive disorder (MDD), the most prevalent mental illness, is a chronic and recurrent condition (Lucas et al., 2011). MDD seems to be associated with abnormalities in regions that mediate emotional and stress responses (Manji et al., 2001). A strong link between suicide and depression has been showed, with more than 86% of suicide victims having a depressive disorder, and committing suicide most often after a major depressive episode (Coryell & Young, 2005; Rihmer, 2007). So, it is possible to study the brain tissue from suicide completers, in order to understand the neurobiological basis of depression. Suicide has been identified as a serious public health problem. Risk factors, such as biological, psychiatric and cultural, interact in a complex manner to this pathology (Bertolote & Fleischmann, 2005). Adenosine is an endogenous nucleoside that influences many functions in the central nervous system (CNS) (Fredholm et al., 2005), acting as a homeostatic modulator and also as a neuromodulator at the synaptic level, where it modulates the release of neurotransmitters, the post-synaptic responsiveness and the action of other receptor systems (Cunha, 2001). Adenosine acts via activation of four G-protein coupled receptors (GPCRs) (Fredholm, 1997), in the brain, it acts especially through activation of two adenosine receptors (ARs), inhibitory A1 (A1R) and facilitatory A2A (A2AR) receptors (Cunha, 2005; Wei et al., 2011). The interest in the role of adenosine in mood disorders arised from previous studies that have recognized a relationship between caffeine intake, mood changes and specific psychiatric symptoms (Kawachi et al., 1996; Lara, 2010; Lucas et al., 2011; Lucas et al., 2013), since caffeine has biological effects as competitive antagonist of A1R and A2AR (Chen et al., 2013; Cunha et al., 2008; El Yacoubi et al., 2003; Fredholm et al., 2005; Fredholm, 2007). There is also evidence that different therapeutic strategies used to control mood disorders are related to the adenosine modulation system (Chen et al., 2013; Cunha et al., 2008; El Yacoubi et al., 2001; Gomes et al., 2011). In animal models of manipulation of ARs, there are modified behavioral responses considered relevant for mood in humans (Gomes et al., 2011). The main goal of this study was to understand the differences in the localization, density and function of both A1R and A2AR between control subjects and suicide completers, in brain areas affected by depression. Also, we aimed to comprehend if these alterations are related with synaptic changes. For this purpose, a postmortem study was performed in male subjects who died by suicide, aged-matched with nonsuicide controls who died by natural causes or accidents. Human brain samples were obtained at autopsies performed in Instituto Nacional de Medicina Legal e Ciências Forenses, I. P. (INMLCF), Coimbra, Portugal. Several brain areas were studied: Brodmann area 25 (BA25); medial caudate nucleus (MC); posterior caudate nucleus (PC) and hippocampus. To comprehend the normal localization of A1R and A2AR we compared the relative abundance of A1R and A2AR in total extracts (TE) and in nerve terminals (NT), isolated using an adapted discontinuous Percoll gradient protocol (Dunkley et al., 1986, 2008), from human brain areas of the same sample. In all brain areas studied, both receptors were found enriched in NT. We then refined the information on ARs subsynaptic localization, using a fractionation method (Phillips et al., 2001; Rebola et al., 2005), and it was observed that A2AR are mainly located outside the active zone and A1R are present in all synaptic fractions. Synaptic changes present in the brain of suicide completers were then studied and it was observed a down-regulation of synaptosomal-associated protein 25 (SNAP25) on BA25 and MC together with a decrease in postsynaptic density protein 95 (PSD95) levels on PC and hippocampus. Astrocytic marker glial fibrillary acidic protein (GFAP) down-regulation was observed in BA25 and hippocampus. Several alterations in the density of ARs, in the different brain regions, were observed in suicide completers when compared with age-matched controls. We described: an up-regulation of A2AR in TE, on BA25 and PC; an up-regulation of A1R in TE, on MC; a down-regulation of A1R in NT, on PC. Due to their particular distribution in the brain and their functional properties, ARs constitute an attractive opportunity for developing innovative compounds for the treatment of specific neurodegenerative and psychiatric disorders, such as MDD. Our work has provided information about changes present in the brains of suicide completers and might contribute to better understand the modulatory role of ARs in depression. There are still numerous questions that demand careful attention to further explore this system and develop novel strategies to control mood disorders, particularly MDD.
A perturbação depressiva major (MDD), a mais predominante das doenças mentais, é uma condição crónica e recorrente (Lucas et al., 2011). A MDD parece estar associada com alterações nas regiões que medeiam as respostas emocionais e de stress (Manji et al., 2001). Uma forte ligação entre o suicídio e a depressão foi demonstrada, uma vez que mais de 86% das vítimas de suicídio apresentavam uma perturbação depressiva, na maioria dos casos, ocorrendo o suicídio depois de um episódio depressivo major (Coryell et al., 2005; Rihmer, 2007). Assim, o tecido cerebral de vítimas de suicídio pode ser utilizado para estudar a neurobiologia da depressão. O suicídio foi identificado como um problema sério de saúde pública. Vários fatores de risco biológicos, psiquiátricos e culturais interagem de uma maneira complexa para esta patologia (Bertolote & Fleischmann, 2005). A adenosina é um nucleósido endógeno que influencia muitas funções do sistema nervoso central (CNS), agindo como modulador homeostático e também como neuromodulador ao nível sináptico, onde modula a libertação de neurotransmissores, a capacidade de resposta pós-sináptica e a ação de outros sistemas receptores (CNS) (Cunha, 2001; Fredholm et al., 2005). A adenosina actua em quatro receptores acoplados à proteína G (GPCRs) (Fredholm, 2007), no cérebro age especialmente através da activação de dois receptores de adenosina (ARs), inibitório A1 (A1R) e facilitatório A2A (A2AR) receptores (Cunha, 2005; Wei et al., 2011). O interesse do papel da adenosina nos distúrbios de humor surgiu de numerosos estudos que reconheceram uma relação entre o consumo de cafeína, alterações de humor e sintomas psiquiátricos específicos (Kawachi et al., 1996; Lara, 2010; Lucas et al., 2011; Lucas et al., 2013), uma vez que a cafeína tem efeitos biológicos como antagonista competitivo dos A1R e A2AR (Chen et al., 2013; Cunha et al., 2008; El Yacoubi et al., 2003; Fredholm et al., 2005; Fredholm, 2007). Também há evidências de que as estratégias terapêuticas utilizadas para controlar distúrbios de humor estão relacionados com o sistema modulatório de adenosina (Chen et al., 2013; Cunha et al., 2008; El Yacoubi et al., 2001; Gomes et al., 2011). Em modelos animais de manipulação de ARs há respostas comportamentais consideradas relevantes para o humor nos humanos (Gomes et al., 2011). O principal objectivo deste estudo era perceber as diferenças na localização, densidade e função dos A1R e A2AR, entre vítimas de suicídio e controlos, em áreas afectadas pela depressão. Além disso, tentámos compreender se essas alterações estão relacionadas com modificações sinápticas. Para este propósito foi realizado um estudo postmortem em sujeitos do sexo masculino suicidas, pareados com controlos da mesma idade que morreram de causas naturais ou acidentes. As amostras de cérebro humano foram obtidas em autópsias realizadas no Instituto Nacional de Medicina Legal e Ciências Forenses, I. P. (INMLCF), Coimbra, Portugal. Foram estudadas diversas áreas cerebrais: área de Brodmann 25 (BA25); núcleo caudado médio (MC); núcleo caudado posterior (PC) e hipocampo. Para compreender a localização normal dos A1R e do A2AR comparámos a abundância relativa dos ARs em extratos totais (TE) e em terminais nervosos (NT), isolados usando um protocolo adaptado de gradiente de Percoll discontínuo (Dunkley et al., 1986, 2008), das áreas cerebrais da mesma amostra. Em todas as áreas estudadas os dois receptores encontram-se enriquecidos nos NT. De seguida, refinámos a informação acerca da localização subsináptica dos ARs, usando um método de fracciomento (Phillips et al., 2001; Rebola et al., 2005) e foi observado que os A2AR estão maioritariamente localizados fora da zona activa da sinapse e os A1R estão presentes em todas as fracções sinápticas. Foram então estudadas alterações sinápticas presentes no cérebro de suicidas e foi observada uma diminuição de densidade da synaptosomal-associated protein 25 (SNAP-25) na BA25 e no MC juntamente com um decréscimo nos níveis de postsynaptic density protein 95 (PSD-95) no PC e no hipocampo. Foi observado uma diminuição da densidade do marcador astrocítico glial fibrillary acidic protein (GFAP) na BA25 e no hipocampo. Várias alterações na densidade dos ARs, nas diferentes regiões do cérebro, foram observadas nas amostras de suicidas quando comparadas com os controlos. Nós descrevemos: aumento da densidade de A2AR em TE, na BA25 e no PC; aumento de densidade de A1R em TE, no MC; diminuição de densidade de A1R em NT, no PC. Devido à sua particular distribuição no cérebro e as suas propriedades funcionais, os ARs constituem uma oportunidade atrativa para desenvolver compostos inovadores para o tratamento de distúrbios psiquiátricos e neurodegenerativos, como a MDD. O nosso trabalho forneceu informação sobre as mudanças presentes no cérebro de suicidas e pode contribuir para uma melhor compreensão do papel modulador dos ARs na depressão. Há ainda numerosas questões, que requerem atenção cuidadosa, para continuar a explorar este sistema e desenvolver novas estratégias para controlar os distúrbios de humor, particularmente, a MDD.

Parkinson’s disease (PD) is a neurodegenerative disorder that is characterised by a reduction of dopamine concentration in the striatum due to degeneration of dopaminergic neurons in the substantia nigra. Currently, first line treatment of PD includes the use of dopamine precursors, dopamine agonists and inhibitors of enzymatic degradation of dopamine, in an effort to restore dopamine levels and/or its effects. However, all these therapeutic strategies are only symptomatic and unfortunately do not slow, stop or reverse the progression of PD. From the discovery of adenosine A2A receptor-dopamine D2 receptor heteromers and the antagonistic interaction between these receptors, the basis of a new therapeutic approach towards the treatment of PD emerged. Adenosine A2A receptor antagonists have been shown to decrease the motor symptoms associated with PD, and are also potentially neuroprotective. The possibility thus exists that the administration of an adenosine A2A antagonist may prevent further neurodegeneration. Furthermore, the antagonism of adenosine A1 receptors has the potential of treating cognitive deficits such as those associated with Alzheimer's disease and PD. Therefore, dual antagonism of adenosine A1 and A2A receptors would be of great benefit since this would potentially treat both the motor as well as the cognitive impairment associated with PD. The affinities (Ki-values between 0.6 mM and 38 mM) of a series of 1,4-dihydropyridine derivatives were previously illustrated for the adenosine A1, A2A and A3 receptor subtypes by Van Rhee and co-workers (1996). These results prompted this pilot study, which aimed to investigate the potential of the structurally related 3,4-dihydropyrimidin-2(1H)-ones (dihydropyrimidones) and 2-amino-1,4-dihydropyrimidines as adenosine A1 and A2A antagonists. In this pilot study, a series of 3,4-dihydropyrimidones and 2-amino-1,4-dihydropyrimidines were synthesised and evaluated as adenosine A1 and A2A antagonists. Since several adenosine A2A antagonists also exhibit MAO inhibitory activity, the MAO-inhibitory activity of selected derivatives was also assessed. A modified Biginelli one pot synthesis was used for the preparation of both series of compounds under solvent free conditions. A mixture of a β- diketone, aldehyde and urea/guanidine hydrochloride was heated for an appropriate time to afford the desired compounds in good yields. MAO-B inhibition studies comprised of a fluorometric assay where kynuramine was used as substrate. A radioligand binding protocol described in literature was employed to investigate the binding of the compounds to the adenosine A2A and A1 receptors. The displacement of N-[3H]ethyladenosin-5’-uronamide ([3H]NECA) from rat striatal membranes and 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) from rat whole brain membranes, was used in the determination of A2A and A1 affinity, respectively. The results showed that both 3,4-dihydropyrimidones and 2-amino-1,4-dihydropyrimidines had weak adenosine A2A affinity, with the p-fluorophenyl substituted dihydropyrimidone derivative (1h) in series 1, exhibiting the highest affinity for the adenosine A2A receptor (28.7 μM), followed by the p-chlorophenyl dihydropyrimidine derivative (2c) in series 2 (38.59 μM). Both series showed more promising adenosine A1 receptor affinity in the low micromolar range. The p-bromophenyl substituted derivatives in both series showed the best affinity for the adenosine A1 receptor with Ki-values of 7.39 μM (1b) and 7.9 μM (2b). The pmethoxyphenyl dihydropyrimidone (1d) and p-methylpneyl dihydropyrimidine (2e) derivatives also exhibited reasonable affinity for the adenosine A1 receptor with Ki-values of 8.53 μM and 9.67 μM, respectively. Neither the 3,4-dihydropyrimidones nor the 2-amino-1,4- dihydropyrimidines showed MAO-B inhibitory activity. Comparison of the adenosine A2A affinity of the most potent derivative (1h, Ki = 28.7 μM) from this study with that of the previously synthesised dihydropyridine derivatives (Van Rhee et al., 1996, most potent compound had a Ki = 2.74 mM) reveals that an approximate 100- fold increase in binding affinity for A2A receptors occurred. However, KW6002, a known A2A antagonist, that has already reached clinical trials, has a Ki-value of 7.49 nM. The same trend was observed for adenosine A1 affinity, where the most potent compound (1b) of this study exhibited a Ki-value of 7.39 μM compared to 2.75 mM determined for the most potent dihydropyridine derivatives (Van Rhee et al., 1996). N6-cyclopentyladenosine (CPA), a known adenosine A1 agonist that was used as a reference compound, however had a Kivalue of 10.4 nM. The increase in both adenosine A1 and A2A affinity can most likely be ascribed to the increase in nitrogens in the heterocyclic ring (from a dihydropyridine to a dihydropyrimidine) since similar results were obtained by Gillespie and co-workers in 2009 for a series of pyridine and pyrimidine derivatives. In their case it was found that increasing the number of nitrogens in the heterocyclic ring (from one to two nitrogen atoms for the pyridine and pyrimidine derivatives respectively) increased affinity for the adenosine A2A and adenosine A1 receptor subtypes, while three nitrogen atoms in the ring (triazine derivatives) were associated with decreased affinity. It thus appears that two nitrogen atoms in the ring (pyrimidine) are required for optimum adenosine A1 and A2A receptor affinity. The poor adenosine A2A affinity exhibited by the compounds of this study can probably be attributed to the absence of an aromatic heterocyclic ring. The amino acid, Phe-168 plays a very important role in the binding site of the A2A receptor, where it forms aromatic - - stacking interactions with the heterocyclic aromatic ring systems of known agonists and antagonists. Since the dihydropyrimidine ring in both series of this pilot study was not aromatic, the formation of aromatic - -stacking interactions with Phe-168 is unlikely. In conclusion, the 3,4-dihydropyrimidone and 2-amino-1,4-dihydropyrimidine scaffolds can be used as a lead for the design of novel adenosine A1 and A2A antagonists, although further structural modifications are required before a clinically viable candidate will be available as potential treatment of PD.
MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Adenosine receptors that belong to the rhodopsin-like G protein-coupled receptors (GPCRs) are involved in a lot of regulatory processes and are widely distributed throughout the body which makes them an attractive target for drugs. However, pharmacological knowledge of these receptors is still limited. A big advance regarding the structural knowledge of adenosine receptors was the development of the first crystal structure of the adenosine A2A receptor in 2008. The crystal structure revealed the amino acids that form the ligand binding pocket of the receptor and depicted the endpoint of receptor movement in the ligand binding process. Within the scope of this work two members of the adenosine receptor family were investigated, namely the adenosine A1 and the A2A receptor (A1R, A2AR). A1R was generated on base of the previously developed A2AR. Receptors were tagged with fluorophores, with the cyan fluorescent protein (CFP) at the C-terminal end of receptor and the Fluorescein Arsenical Hairpin binder (FlAsH) binding sequence within the third intracellular loop of receptors. Resulting fluorescent receptor sensors A1 Fl3 CFP and A2A Fl3 CFP were investigated with help of Fluorescence Resonance Energy Transfer (FRET) measurements within living cells. FRET experiments enable the examination of alteration in the distance of two fluorophores and thus the observation of receptor dynamical movements. For comparison of A1R and A2AR regarding receptor dynamical movement upon ligand binding, fluorescent receptor sensors A1 Fl3 CFP and A2A Fl3 CFP were superfused with various ligands and the outcomes of FRET experiments were compared regarding signal height of FRET ratio evoked by the distinct ligand that is correlated to the conformational change of receptor upon ligand binding. Beside the different direction of FRET ratio upon ligand binding at A1R and A2AR sensor, there were differences observable when signal height and association and dissociation kinetics of the various ligands investigated were compared to each other. Differences between the adenosine receptor subtypes were especially remarkable for the A1R subtype selective agonist CPA and the A2AR subtype selective agonist CGS 21680. Another part of the project was to investigate the influence of single amino acids in the ligand binding process within the fluorescent A1R sensor. Amino acid positions were derived from the crystal structure of the A2AR forming the ligand binding pocket and these amino acids were mutated in the A1R structure. Investigation of the A1R sensor and its mutants regarding confocal analysis showed involvement of some amino acids in receptor localization. When these amino acids were mutated receptors were not expressed in the plasma membrane of cells. Some amino acids investigated were found to be involved in the ligand binding process in general whereas other amino acids were found to have an influence on the binding of distinct structural groups of the ligands investigated. In a further step, A1R and A2AR were N-terminally tagged with SNAP or CLIP which allowed to label receptor sensors with multiple fluorophores. With this technique receptor distribution in cells could be investigated with help of confocal analysis. Furthermore, ligand binding with fluorescent adenosine receptor ligands and their competition with help of a non-fluorescent antagonist was examined at the SNAP tagged A1R and A2AR. Finally the previously developed receptor sensors were combined to the triple labeled receptor sensors SNAP A1 Fl3 CFP and SNAP A2A Fl3 CFP which were functional regarding FRET experiments and plasma membrane expression was confirmed via confocal analysis. In the future, with the help of this technique, interaction between fluorescent ligand and SNAP tagged receptor can be monitored simultaneously with the receptor movement that is indicated by the distance alteration between FlAsH and CFP. This can lead to a better understanding of receptor function and its dynamical movement upon ligand binding which may contribute to the development of new and more specific drugs for the A1R and A2AR in the future
Adenosin Rezeptoren, die zur Gruppe der Rhodopsin-ähnlichen G Protein-gekoppelten Rezeptoren (GPCRs) gehören, sind in eine Vielzahl regulatorischer Prozesse eingebunden und weit im Körper verbreitet. Das macht sie zu einer interessanten Zielstruktur für Arzneistoffe. Das Wissen über die Struktur der Adenosin Rezeptoren ist jedoch noch begrenzt. Ein großer Fortschritt zu mehr strukturellem Wissen war die Entwicklung der ersten Kristallstruktur des Adenosin A2A Rezeptors im Jahr 2008. Mit der Kristallstruktur wurden die Aminosäuren bekannt, die die Ligandenbindetasche dieses Rezeptors formen. Zudem gab die Kristallstruktur Einblick in den Endpunkt der dynamischen Rezeptorbewegung nach Ligandenbindung. Im Rahmen der hier vorgestellten Arbeit wurden zwei Mitglieder der Adenosin Rezeptor Familie, der Adenosin A1 Rezeptor und der Adenosin A2A Rezeptor (A1R, A2AR), genauer untersucht. Der A1R wurde auf Basis des vor kurzem veröffentlichten A2AR entwickelt. Die Rezeptoren wurden mit Fluorophoren versehen, zum einen mit dem cyan fluoreszierenden Protein (CFP) am C-Terminus des Rezeptors und zum anderen mit der Bindesequenz des kleinen Fluorophors "Fluorescein Arsenical Hairpin binder" (FlAsH) in der dritten intrazellulären Schleife des Rezeptors. Die daraus resultierenden Rezeptorsensoren A1 Fl3 CFP und A2A Fl3 CFP wurden mit Hilfe des Fluoreszenz Resonanz Energie Transfers (FRET) in lebenden Zellen erforscht. FRET Messungen ermöglichen es, eine Änderung der Distanz zwischen den beiden Fluorophoren und damit Rezeptorbewegungen zu untersuchen. Um A1R und A2AR bezüglich dynamischer Rezeptorbewegungen nach Ligandenbindung vergleichen zu können, wurden die fluoreszierenden Rezeptorsensoren A1 Fl3 CFP und A2A Fl3 CFP mit verschiedenen Liganden umspült. Die Ergebnisse der FRET Messungen bezüglich ihrer Höhe des FRET Ratio wurden verglichen, welche mit der Konformationsänderung des Rezeptors nach Ligandenbindung zusammenhängt. Neben der unterschiedlichen Richtung des FRET Ratio nach Ligandenbindung am A1R und A2AR Sensor waren Unterschiede bezüglich der Signalhöhe und der Bindungs- und Dissoziationskinetiken feststellbar, wenn die verschiedenen Liganden miteinander verglichen wurden. Unterschiede zwischen den Adenosin Rezeptor Subtypen waren speziell für den A1R subtypselektiven Agonist CPA und für den A2AR subtypselektiven Agonist CGS 21680 feststellbar. Einen weiteren Punkt in diesem Projekt stellte die Erforschung des Einflusses, den einzelne Aminosäuren im fluoreszierenden A1R Sensor auf den Prozess der Ligandenbindung haben, dar. Die Position der Aminosäuren wurde der Kristallstruktur des A2AR entnommen und entsprechende Aminosäuren im A1R mutiert. Die konfokalmikroskopische Analyse des A1R Sensors und seiner Mutanten ergab, dass einige Aminosäuren direkt an der zellulären Expression des Rezeptors beteiligt waren. Wurden diese Aminosäuren mutiert, wurde der Rezeptor nicht in der Plasmamembran der Zellen exprimiert. Einige Aminosäuren die untersucht wurden,hatten einen generellen Einfluss auf die Bindung der Liganden, andere Aminosäuren hatten mehr Einfluss auf die Bindung bestimmter struktureller Gruppen der untersuchten Liganden. In einem weiteren Schritt wurden A1R und A2AR am N-terminalen Rezeptorende mit SNAP oder CLIP versehen, was eine Markierung der Rezeptoren mit einer Vielzahl an Fluorophoren erlaubt. Mit Hilfe dieser Technik konnte die Verteilung der Rezeptoren in der Zelle mit konfokaler Mikroskopie untersucht werden. Des Weiteren wurde die Bindung von fluoreszierenden Adenosin Rezeptor Liganden und deren Verdrängung mit einem nicht-fluoreszierenden Adenosin Rezeptor Antagonist erforscht. Am Ende des Projekts wurden die zuvor beschriebenen fluoreszierenden Rezeptorsensoren zu dreifach fluorophormarkierten Rezeptorsensoren kombiniert, was zu den Sensoren SNAP A1 Fl3 CFP und SNAP A2A Fl3 CFP führte. Beide Rezeptorsensoren waren funktionell bezüglich FRET Experimenten und der Expression in der Plasmamembran der Zellen. In Zukunft können mit dieser Methode gleichzeitig die Bindung von fluoreszierenden Liganden am SNAP-markierten Rezeptor, so wie die Rezeptorbewegung beobachtet werden, die durch eine Distanzänderung zwischen CFP und FlAsH angezeigt wird. Das kann zu einem besseren Verständnis der Rezeptorfunktion und der dynamischen Rezeptorbewegung nach Ligandenbindung führen, die in Zukunft zur Entwicklung spezifischerer Wirkstoffe am A1R und A2AR beitragen könnte

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2012
A Esclerose Lateral Amiotrópica (ELA) é uma doença progressiva e fatal caracterizada pela degeneração selectiva dos neurónios motores do córtex motor, tronco cerebral e medula espinal, que provoca atrofia muscular, paralesia e morte por falha respiratória. A etiologia da doença continua desconhecida, mas com um consenso de que o dano dos neurónios motores é causado por uma rede de processos patológicos complexos. Os mecanismos envolvidos na degeneração dos neurónios motores são melhor conhecidos num subtipo da doença causada por mutações na enzima superóxido dismutase 1 (SOD1). Esta enzima actua na eliminação de radicais livres de oxigénio e na ELA o processo de degeneração neuronal deve-se a um ganho de função da SOD1. A adenosina tem uma função importante na modulação da transmissão sináptica no SNC e SNP, actuando a dois níveis: inibitório, modulado pelos receptores do subtipo A1 e excitatório, mediado pelos receptores do subtipo A2A. É conhecido que a expressão dos receptores A1 e A2A da adenosina está alterada nalgumas doenças neurodegenerativas, mas o seu papel na ELA é ainda muito pouco conhecido. O objectivo deste trabalho foi determinar o efeito da ELA na expressão proteica e de mRNA dos receptors A1 e A2A da adenosina no decurso da doença. O modelo de murganhos transgénicos para o gene SOD1 humano com a mutação G93A foi usado neste trabalho. Os níveis proteicos e de mRNA de ambos os receptores foram quantificados através das técnicas de immunoblotting e PCR quantitativo em tempo real, respectivamente. Foram estudados diferentes tecidos do SNC e SNP, nomeadamente, córtex e medula espinal (apenas immunoblotting) e nervo frénico-diafragama, de animais selvagens e portadores da doença nas fases pre-sintomática (4-6 semanas) e sintomática (13-14 semanas). Resultados deste estudo indicaram níveis proteicos não alterados nos SNC e SNP do receptor A1 ao longo da progressão da doença. No entanto, observou-se uma sobreexpressão dos receptores A2A no córtex na fase pre-sintomática e um decréscimo na fase sintomática. Os outros tecidos mantiveram-se inalterados no que se refere aos receptores A2A em ambas as fases da doença. A avaliação da expressão de mRNA no diafragma não revelou quaisquer alterações em ambos os receptores da adenosina durante a progressão da doença. Assim, no que se refere aos receptores da adenosina em ELA, as primeiras alterações parecem ocorrer logo no início da doença nos receptores A2A do SNC.
Amyothrophic Lateral Sclerosis (ALS) is a progressive and fatal disease categorized by a selective degeneration of motor neurons from the cerebral cortex, brainstem and spinal cord that provokes muscle atrophy, progressive paralysis and death due to respiratory failure. The etiology of most ALS cases remains unknown but there is a current consensus that motor neuron degeneration is caused by a complex interaction between multiple pathogenic processes. The mechanisms of motor neuron degeneration are best understood in the subtype of disease caused by mutations in the enzyme superoxide dismutase 1. This enzyme is enrolled in the degradation of free oxygen radicals and in ALS neuronal damage is due to its gain-of-function. Adenosine has a central role as a neuromodulator of the CNS and PNS synaptic transmission. Adenosine acts at two levels: inhibitory through the subtype A1 receptor and excitatory through the subtype A2A receptor. Variation on the expression of A1 and A2A receptors has been identified in some neurodegenerative diseases, but their role in ALS is not yet understood. The objective of this work was to determine the effect of ALS on the protein and mRNA expression of A1 and A2A adenosine receptors through disease progression. The transgenic model of mice carrying the human SOD1 gene with the G93A mutation was used in this work. Protein and mRNA levels of both receptors were quantified through immunblotting and quantitative real time PCR, respectively. Different tissues of the CNS and PNS, namely cortex and spinal cord (immunoblotting only) and phrenic nerve-diaphragm were studied in wild-type and transgenic mice in the pre-symptomatic (4-6 weeks) and symptomatic (13-14 weeks) phases of the disease. Results from this study indicate unaltered A1 receptor protein levels at the CNS and PNS through disease progression. However, there is an overexpression of A2A receptors in the cortex of pre-symptomatic mice and a decrease in the symptomatic phase. The A2A receptors are unaltered in the other tissues in both phases of the disease. The mRNA evaluation does not reveal significant alterations in both adenosine receptors during disease progression. Thus, regarding adenosine receptors in ALS, the first changes seem to occur early in the disease at the CNS in A2A receptors.

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.

博士
國立陽明大學
生理學研究所
95
Abstract The effect of adenosine on the fEPSP was examined in lateral olfactory tract (Ia input) and associative tract (Ib input) in rat piriform cortex. The fEPSP evoked in Ia input showed paired-pulse (PP) facilitation, and in Ib input it showed PP depression, suggesting a lower resting release probability in the Ia input. This was supported by results showing that MK801 blocked the NMDA-receptor fEPSP at a faster rate in the Ib input. Adenosine caused a dose-dependent inhibition of the fEPSP in both inputs, the sensitivity being higher in the Ib input. This effect was mimicked by the A1 receptor agonist, CHA, and antagonized by co-application of the antagonist, DPCPX, showing that adenosine was acting at A1 receptors. Application of DPCPX alone increased the fEPSP, the increase being larger in the Ia input. DPCPX also caused PP depression in both inputs, and the PP ratios measured in its presence were very similar in both inputs. These results suggest a lower endogenous concentration of adenosine in the Ib sublayer than the Ia sublayer, which might account for the native difference in the resting release probability of the two inputs. The inhibition of the fEPSP in both inputs by adenosine was associated with an increase in the PP ratio and with significant lowering of the MK801 blocking rate of the NMDA-receptor fEPSP, suggesting a presynaptic location of the A1 receptors. Blocking of N-, P/Q-type calcium channels occluded the inhibition by adenosine, indicating a downregulation of presynaptic calcium channels by A1 receptors.

by Zhen Yang.
On t.p. "1" is subscript following A in the title.
Thesis (Ph.D.)--Chinese University of Hong Kong, 1992.
Includes bibliographical references (leaves 138-139).
Acknowledgements --- p.i
List of Nomenclature --- p.ii-ix
"Abstracts (for Chapter 2,Chapter 3,and Chapter 4)" --- p.x
Chapter Chapter 1. --- General Introduction --- p.1-8
Chapter Chapter 2. --- Synthesis of 5-(3-Hydroxypropyl)-7-methoxy-2-(3'-methoxy-4'- hydroxyphenyl)-benzo[b]furan-3-carbaldehyde (XH14) (18)
Chapter 2.1. --- Introduction --- p.9-12
Chapter 2.2. --- Isolation and Structure Elucidation of XH14 (18)
Chapter 2.2.1. --- Material and method --- p.13-17
Chapter 2.2.2. --- Structure elucidation --- p.18-23
Chapter 2.3. --- Results and Discussion
Chapter 2.3.1. --- Total synthesis of XH14 (18) --- p.24-52
Chapter 2.3.2. --- Chemical modification of XH14 (18)
Chapter (a) --- Synthesis of 3-hydroxymethyl-5-(3-hydroxypropyl)- 7-methoxy-2-(3'-methoxy-4'-hydroxy phenyl)-benzo[b] furan(49) and 3-hydroxymethyl-5-(2-methoxycarbonyl ethyl)-7-methoxy-2-(3'-methoxy-4'-hydroxy phenyl)- benzo[b]furan (66) --- p.53-54
Chapter (b) --- Synthesis of 5-(2-carboxyethyl)-7-methoxy-2-(3'- methoxy-4'-hydroxyphenyl)-benzo[b]furan-3- carbaldehyde (67) --- p.54
Chapter (c) --- Synthesis of 5-(3-Hydroxypropyl)-7-methoxy-3- methyl-2-(3'-methoxy-4'-hydroxyphenyl)- benzo[b]furan (68) and 5-(2-methoxycarbonylethyl)-7- methoxy-3-methyl-2-(3'-methoxy-4'-hydroxyphenyl)- benzo[b]furan (69) --- p.54-55
Chapter (d) --- Synthesis of 5-(2-carboxy-trans-ethenyl)-7- methoxy-2-(3'-methoxy-4'- hydroxy phenyl) benzo[b]furan-3-carbaldehyde (70) --- p.55-56
Chapter (e) --- Synthesis of 4-bromo-5-(3-hydroxypropyI)-7- methoxy-2-(3'-methoxy-4'- hydroxy phenyl)- benzo[b]furan-3-carbaldehyde (72) --- p.56-57
Chapter (f) --- Synthesis of 4-acetyl-5-(3-hydroxypropyl)-7- methoxy-2-(3'-methoxy-4'-hydroxyphenyl)-benzo [b]furan (73) and 3-acetyI-5-(3-hydroxypropyl)-7- methoxy-2-(3'-methoxy-4'-hydroxy phenyl)- benzo[b]furan (75) --- p.57-61
Chapter (g) --- Synthesis of 3-nitro-5-(3-hydroxypropyl)-7- methoxy -2-(3'-methoxy-4'-hydroxyphenyl)- benzo[b]furan (77) and 4-nitro-5-(3-hydroxyphenyl)-7- methoxy-2-(3'-methoxy-4'-hydroxyphenyl)- benzo[b]furan (78) --- p.61-64
Chapter (h) --- Synthesis of 3-(α-hydroxyethyl)-5-(3-hydroxy propyl) -7-methoxy-2- (3'-methoxy-4' -hydroxyphenyl) - benzo[b]furan (81) --- p.64
Chapter (i) --- Synthesis of 5-(3-hydroxypropyl)-7-methoxy-2-(3'- methoxy-4'-benzyloxyphenyl )-benzo[b]furan-4- carbaldehyde (82) --- p.64
Chapter 2.4. --- Structure-activity Relationship of A1 Antagonists --- p.65-71
Chapter 2.5. --- Conclusion --- p.72
Chapter 2.6. --- Experimental Section --- p.73-97
Chapter 2.7. --- References --- p.98-103
Chapter Chapter 3. --- "Synthesis of 9,10-Dihydro-10,10-dimethoxyanthracen-9-one"
Chapter 3.1. --- Introduction --- p.104
Chapter 3.2 --- Results and Discussion --- p.105-118
Chapter 3.3. --- Experimental Section --- p.119-124
Chapter 3.4. --- References --- p.125-128
Chapter Chapter 4. --- "Synthesis of 10,11-Dimethoxydibenz[b,f]oxep in"
Chapter 3.1. --- Introduction --- p.129
Chapter 3.2. --- Results and Discussion --- p.130-133
Chapter 3.3. --- Experimental Section --- p.134-137
Chapter 3.4. --- References --- p.138-139
Appendix
Spectra --- p.140-145

Dissertação de mestrado em Biologia Celular e Molecular apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.
Os receptores A2a da adenosina são actualmente considerados um alvo terapêutico na doença de Parkinson pois, devido à co-localização e interacção funcional com os receptores D2 da dopamina nos neurónios do estriado, a modulação com antagonistas A2a tem um efeito compensatório do défice de dopamina causado pela degeneração dos neurónios dopaminérgicos que projectam da substantia nigra para o estriado, melhorando a disfunção motora. Por outro lado, os antagonistas dos receptores A2a também mostraram efeitos neuroprotectores contra a degeneração dopaminérgica induzida por toxinas em modelos animais da doença de Parkinson, um efeito atribuído à inibição de mecanismos neuro-inflamatórios. No entanto, a activação dos receptores A2a é requerida para alguns efeitos do GDNF (Glial cell line-Derived Neurotrophic Factor), um factor neurotrófico amplamente reconhecido como protector e indutor da regeneração dos neurónios dopaminérgicos. O nosso grupo mostrou recentemente que a lesão selectiva dos neurónios dopaminérgicos em culturas de células da substantia nigra pode induzir o aumento da expressão de GDNF em astrócitos. No presente estudo utilizámos um rato modelo da doença de Parkinson, induzido pela injecção de 6-hidroxidopamina (6-OHDA) no estriado, para investigar a relação entre receptores da adenosina, expressão de GDNF e protecção dos neurónios dopaminérgicos. Em estudos de imunohistoquímica após a lesão da via nigra-estriatal, observámos um aumento significativo da expressão dos receptores A2a em astrócitos no estriado, particularmente em astrócitos que proliferaram nas zonas de transição entre zonas lesadas e zonas não afectadas. A observação, também nestas zonas, do aumento da expressão da tirosina hidroxilase (TH), um marcador dos terminais dopaminérgicos, sugere uma relação entre aumento dos receptores A2a e sobrevivência dos neurónios dopaminérgicos, e não apoia a ideia de que a activação dos receptores A2a em situações de lesão é deletéria para a sobrevivência dosneurónios dopaminérgicos. No entanto, em estudos preliminares em culturas de células da substantia nigra, observámos que a inibição dos receptores A2a teve efeitos protectores na sobrevivência dos neurónios dopaminérgicos contra a toxicidade da 6-OHDA. Nos estudos in vivo não observámos aumento da expressão de GDNF, avaliados por Western blot em extractos proteicos do estriado. No rato modelo da doença de Parkinson, observámos também um aumento marcado dos receptores A1 da adenosina no estriado, em terminais dopaminérgicos e em corpos celulares que expressam TH. A aquisição de fenótipo dopaminérgico por neurónios GABAérgicos do estriado lesado foi descrito por outros investigadores, e o nosso trabalho sugere que estes neurónios expressam também receptores A1 da adenosina. Observámos também que a degeneração da via nigra-estriatal está associada ao aumento da expressão dos receptores A1 na substantia nigra em neurónios não-dopaminérgicos cujos corpos celulares estão segregados dos corpos celulares dopaminérgicos nas regiões dendríticas da substantia nigra pars reticulata. Em conclusão, os nossos resultados num modelo animal da doença de Parkinson mostram que a lesão dos neurónios dopaminérgicos pode induzir o aumento da expressão dos receptores A2a da adenosina em astrócitos, e dos receptores A1 em terminais dopaminérgicos no estriado, em zonas de transição da lesão, onde parecem ocorrer tentativas de reparação evidenciadas pelo aumento da expressão da TH. Embora os resultados não provem uma relação entre a expressão dos receptores da adenosina e protecção dos neurónios dopaminérgicos, os nossos resultados não apoiam a ideia de que a activação dos receptores A2a contribua para a degeneração dopaminérgica.

Indiana University-Purdue University Indianapolis (IUPUI)
Adenosine is widely thought to elicit coronary vasodilation and attenuate smooth muscle cell (SMC) proliferation, thereby providing cardioprotection. We cloned the porcine adenosine A1 receptor (A1R) subtype and found that it paradoxically stimulated proliferation of cultured coronary SMC by the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) signaling pathways, thus suggesting A1R dysregulation could play a role in coronary artery disease (CAD), restenosis, and regulation of coronary blood flow (CBF). We utilized the Ossabaw swine model of metabolic syndrome (MetS) to test the hypothesis that A1R activation contributes to development of CAD, in-stent stenosis, and CBF regulation. Swine were fed standard chow (Lean) or excess calorie atherogenic diet for over 20 weeks, which elicited MetS characteristics and coronary atherosclerosis compared to Lean. We observed increased A1R in native CAD in MetS, which was reversed by exercise training, and upregulation of A1R expression and A1R-ERK1/2 activation in an in vitro organ culture model of CAD. Intracoronary stent deployment followed by different durations of recovery showed A1R upregulation occurred before maximal in-stent stenosis in vi vivo. More importantly, selective A1R antagonism with 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX)-eluting stents decreased coronary ERK1/2 activation and reduced in-stent stenosis comparable to Taxus® (paclitaxel-eluting stents). A1R antagonism potentiated vasodilatory effects of some vasodilators other than adenosine in porcine coronary microcirculation under basal conditions. Short-term exercise training around stenting prevented stent-induced microvascular dysfunction and attenuated native atheroma in the genetically lean Yucatan swine. Conclusions: A1R upregulation and activation contributes to coronary in-stent stenosis in vivo in MetS, plays a role in the development of coronary atherosclerosis in vitro, and might involve in CBF dysregulation in dyslipidemia and stenting. Exercise training decreased A1R expression in atherosclerosis, reduced native atheroma, and prevented stent-induced microvascular dysfunction. Selective pharmacological antagonism of A1R holds promise for treatment of CAD.

Tese de doutoramento em Biociências, na especialidade de Neurociências, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra
Este trabalho foi iniciado partindo da premissa em como, no cérebro, a glucose disponível regula a memória e a cognição em humanos saudáveis e pacientes com demência. Para além disso, a desregulação da glucose cerebral tem sido associada a doenças neuropsiquiátricas e as alterações na captação de glucose em áreas cerebrais específicas são considerados marcadores seletivos no diagnóstico de doenças neuropsiquiátricas. O trabalho experimental apresentado nesta tese de doutoramento teve como objetivo revelar como os neuromoduladores, incluindo a insulina, regulam o metabolismo da glicose no cérebro e quais as consequências dos danos ao nível do metabolismo da glicose cerebral em modelos animais. Para concretizar este objectivo, efetuamos estudos bioquímicos e eletrofisiológicos em estruturas cerebrais que se correlacionam com a cognição e personalidade, que são, o hipocampo e o córtex frontal. Também procedemos a uma bateria de testes comportamentais em roedores. Primeiro, optimizamos vários métodos in vitro para medições espaço-temporais e quantitativas da captação e metabolismo da glucose em fatias de hipocampo, fatias do córtex frontal e em culturas celulares. Depois, exploramos as características básicas da captação de glucose e metabolismo em fatias de hipocampo, incluindo o papel das vias de sinalização da insulina e de duas outras importantes famílias de neuromoduladores. Aqui, reportamos que a administração sistémica de streptozotocina (induzindo diabetes tipo 1) afecta tanto o repouso como a despolarização induzida pela captação e metabolismo da glucose dependente do receptor de canabinóides CB1 no hipocampo. Notavelmente, esta área cerebral é responsável pela formação e evocação da memória, que é regulada pela sinalização da insulina. Usando o mesmo composto diabetogénico mas agora administrado intracerebroventricularmente, obtivemos um modelo animal previamente caracterizado como diabetes cerebral, evitando a interferência de modificações periféricas. Além disso, os resultados apontaram para que a disfunção sináptica observada nas experiências de eletrofisiologia tenham ocorrido antes da perda sináptica e coincidiu com a disfunção metabólica. Em seguida, outro modelo animal foi utilizado, associado com disfunção metabólica, um modelo pré-diabético induzido pelo consumo de uma dieta rica em sacarose (Hsu). Este modelo tem a vantagem de ser menos invasivo do que a injeção cerebral de STZ e melhor mimetizar as doenças associadas ao estilo de vida humano, e para além disso permitir ainda o estudo da fase inicial da doença, a fase anterior à ocorrência da diabetes. De acordo com a análise de hipocampo por espectroscopia de elevada resolução por rotação em ângulo mágico (HRMAS) em tecidos de animais Hsu, a dieta Hsu induziu modificações metabólicas periféricas sem um impacto aparente no metabolismo cerebral, apesar destes animais exibirem danos na memória. Procuramos também modificações nos circuitos da via hipocampal Schaffer-CA1, que está conectada com tarefas dependentes de memória hipocampal e não foram detetadas alterações. Posteriormente, questionamos se a dieta Hsu afectaria outra via mediadora da consolidação da memória espacial e o processo de reconhecimento em ratos, nomeadamente a via temporomónica. Observamos que a dieta Hsu danifica a plasticidade sináptica na via temporomonica-CA1 das sinapses piramidais. Adicionalmente, testamos o papel do receptor adenosinérgico nestas vias e observamos que, no hipocampo, a dieta Hsu regula para cima o receptor A1. Seguidamente, investigamos a hipótese de que a ativação do receptor A2B com baixa afinidade para a adenosina pudesse promover a captação de glucose em neurónios e astrócitos relacionando atividade cerebral com o metabolismo energético. Desenvolvemos um protocolo para a medição fluorescente em tempo real da captação de deoxiglucose em fatias de hipocampo, um complemento ideal das análises quantitativas. Verificamos que a ativação do receptor A2B está associada com o aumento tónico e instantâneo do transporte de glucose para os neurónios e astrócitos no cérebro de ratinho, levantando a possibilidade de em futuras investigações se avaliar o potencial clínico deste novo mecanismo glucoregulador. Em conclusão, este trabalho de tese contribuiu para elucidar os mecanismos pouco definidos que regulam o metabolismo da glucose cerebral. Encontramos novas provas que ligam a deficiência da plasticidade sináptica hipocampal e de memória às doenças metabólicas. Também identificamos o receptor A2B como um possível novo alvo terapêutico para estimular o metabolismo da glucose cerebral com o objectivo de atenuar o aparecimento de doenças cerebrais.
This work started from the known premise that, in the brain, glucose availability regulates memory and cognition in both healthy humans and dementia patients. Furthermore, cerebral glucose deregulation has been associated with neuropsychiatric disorders, and alterations in glucose uptake in specific brain areas are selective hallmarks for the diagnosis of neuropsychiatric diseases. The experimental work presented in this doctoral thesis aimed at unveiling how neuromodulators including insulin regulate glucose metabolism in the brain and what are the consequences of impaired brain glucose metabolism in animal models. To this aim, biochemical and electrophysiological studies were performed in brain structures that correlate with cognition and personality, that is, the hippocampus and the frontal cortex. Also a battery of behavioural studies was carried out in laboratory rodents. To begin with, various in vitro methods for both the spatiotemporal and the quantitative measurement of glucose uptake and metabolism in acute hippocampal and frontocortical slices and cell cultures were optimized. Then the basic characteristics of glucose uptake and metabolism in the hippocampal slice were explored, including the role of the insulin signalling pathway and the role of two other important neuromodulator families. We found and report for the first time that systemic streptozotocin-induced untreated type-1 diabetes affects both the resting and depolarization-induced glucose uptake and metabolism in a fashion dependent on the cannabinoid CB1 receptor in the hippocampus. Notably, this brain area is responsible for the formation and recall of memory, which is regulated by insulin signalling. Using the same diabetogenic compound but now intracerebroventricularly administered, we achieved a previously characterized animal model of cerebral diabetes, thus avoiding confounding peripheral changes. This procedure led to the impairment of hippocampusdependent memory. Furthermore, the results pointed out that synaptic dysfunction observed in the electrophysiological experiments occurred before the loss of synapses and coincided with metabolic dysfunction. Next, we moved to another animal model associated with metabolic dysfunction, a prediabetic model induced through the consumption of a high sucrose diet (HSu). This model has the advantage of being less invasive than cerebral STZ-injection, and mimics better human lifestyle-dependent disorders; furthermore it enables the study of the initial phase of the disease, prior to the occurrence of diabetes. According to the highresolution magic angle spinning spectroscopic analysis of the hippocampal tissue, HSuinduced peripheric metabolic modifications did not cause an apparent impact on cerebral metabolism, albeit these HSu animals exhibited memory impairment. We looked for circuitry modifications in the hippocampal Schaffer-CA1 pathway, which is connected to hippocampal-dependent memory task, but none was found. Subsequently, we asked if HSu affected another pathway mediating spatial memory consolidation and recognition process of rats, namely, the temporoammonic branch of the perforant path. We observed that high sucrose consumption impairs synaptic plasticity at the temporoammonic pathway-CA1 pyramidal synapses. Additionally, we tested the role of adenosine receptors in those pathways and observed that high sucrose consumption upregulates adenosine A1R in the hippocampus. Next we investigated the hypothesis that the activation of the low-affinity adenosine A2B receptor (A2BR) could promote glucose uptake in neurons and astrocytes, thereby possibly linking brain activity with energy metabolism. We developed a protocol for real-time fluorescent measurement of deoxyglucose uptake in hippocampal slices, ideal to complement quantitative analyses. We found that A2BR activation is associated with an instant and tonic increase of glucose transport into neurons and astrocytes in the mouse brain. These prompt further investigations to evaluate the clinical potential of this novel glucoregulator mechanism. In conclusion, the present thesis work has contributed to the elucidation the illdefined mechanisms that regulate brain glucose metabolism. We have found additional proofs linking impaired hippocampal synaptic plasticity and memory with metabolic disorders. We also identified the A2BR as a possible novel therapeutic target to boost brain glucose metabolism with the objective of mitigating the outcome of brain disorders.
NARSAD
Santa Casa da Misercórdia
DARPA
Fundação para a Ciência e Tecnologia