Дисертації з теми "Adenosine-signalling"

The release and clearance of adenosine are reasonably well-documented in the mature CNS but relatively little is known about how adenosine signalling changes during postnatal development. The activation of presynaptic A1 receptors (A1R) at cerebellar parallel fibre terminals is known to inhibit synaptic transmission and the expression of A1R has been observed in mature rat cerebellar slices. However its distribution during development or in relation to parallel fibre–Purkinje cell (PF-PC) synapses has not previously been described. In the mature cerebellum blockade of presynaptic A1R at PF-PC synapses enhances synaptic transmission suggesting an inhibitory adenosine tone and an extracellular purine tone is detectable with microelectrode biosensors under basal conditions. The active release of adenosine can be stimulated with trains of activity in the molecular layer of mature slices although this does not appear to be a source of the basal extracellular adenosine tone. This study used immunohistochemistry to determine the distribution of A1R at PFPC synapses in cerebellar slices at postnatal day 3 prior to PF-PC synapse formation, postnatal days 8-14 and postnatal days 21-28. This study also used cerebellar slices from rats at postnatal days 9-14 to investigate the pharmacological profile of the immature rat PF-PC synapse with electrophysiology and microelectrode biosensors. The immunohistochemistry suggests that A1R are widely distributed across Purkinje cell bodies and their dendrites and within the granule layer of the cerebellum and that its expression does not change during development. The same staining patterns were also observed prior to PF-PC synapse formation. Application of adenosine resulted in a variable A1R-mediated inhibition at immature PF-PC synapses. This did not appear to be gender-specific or correlated with age of rat and the synapses otherwise appeared identical in their properties. The comparison of log concentration-response curves generated for an A1R agonist suggested that some A1R may have a lower efficacy at this stage of development. Blockade of presynaptic A1R at immature PF-PC synapses suggested that an inhibitory adenosine tone is low or absent at this stage of development and is not the result of a low A1R expression or developmental differences in A1R efficacy. Inhibition of adenosine clearance via adenosine deaminase, adenosine kinase and equilibrative transporters had little effect on synaptic transmission suggesting that little adenosine is moving between the intracellular and extracellular spaces under basal conditions in immature slices. Active adenosine release measured by electrophysiology and microelectrode biosensors could be stimulated with hypoxia in immature slices but this was delayed and slower in comparison to the release observed in mature slices. Adenosine could not be actively released at immature PFPC synapses in response to electrical stimulation in the molecular layer.

Myocardial fibrosis contributes to the pathogenesis of diverse forms of cardiac hypertrophy. Recent evidence suggests a crucial role for purinergic signalling in the formation of tissue fibrosis. In particular, inhibition of adenosine A2A receptors (A2AR) in animal models for hepatic and skin fibrosis resulted in a reduction of fibrosis formation. The specific hypothesis of this investigation was that myocardial stress results in increased interstitial levels of adenosine, which activate cardiac fibroblasts via adenosine A2AR. Accordingly, the specific objective of this investigation was to delineate the role of adenosine A2AR signalling in the development of myocardial fibrosis. In vitro, isolated cardiac fibroblasts demonstrated a significant increase in collagen secretion into CCM upon specific A2AR stimulation. This stimulatory effect was inhibited by the addition of a specific A2AR inhibitor. In vivo, models for cardiac hypertrophy including the transgenic cardiac actin E99K hypertrophic cardiomyopathy model, and a transverse aortic constriction (TAC) model of pressure overload, were investigated as murine models for myocardial fibrosis (MF). In cardiac actin E99K hearts the occurrence of cardiac fibrosis was associated with ventricular dysfunction, as well as energetic perturbations. In addition, a direct linear correlation between myocardial collagen content and interstitial adenosine levels was found in actin E99K hearts upon microdialysis experiments. Crossbreeding of actin E99K and A2AR knock-out (KO) mice resulted in a significant reduction of myocardial collagen content and fibrosis in E99K heterozygous A2AR KO animals compared to E99K heterozygous A2AR wild-type (WT) animals. Further, adenosine A2AR KO mice undergoing transverse aortic constriction demonstrated significantly less fibrosis formation compared to constricted WT mice. This was associated with a significant rescue of cardiac function. In addition, pharmacologic adenosine A2AR inhibition using the antagonist ZM241385 demonstrated a partial rescue of myocardial fibrosis in both TAC and E99K animals. These data suggest that adenosine the A2AR plays a crucial role in the formation of myocardial fibrosis in a variety of cardiac pathologies and that this pathway is susceptible to pharmacologic modulation. A2AR manipulation may contribute to further understanding of pathophysiological pathways in the development and progression of cardiac disease and represents an excellent therapeutic target for clinically available A2AR antagonists.

African trypanosomiasis is caused by a unicellular eukaryote that parasitizes multicellular organisms and causes medically and economically important diseases in humans (Human African Trypanosomiasis) and their domestic animals (African Animal Trypanosomiasis). Incidence is currently declining due to the application of present chemotherapy, although the drugs are old, toxic, difficult to administer and in some cases expensive, and of diminishing efficacy due to resistance. However, this trend needs to be sustained with the discovery of new compounds active against the resistant strains. These new treatment options must meet the current pharmacological requirements, must be parasite specific and must be relatively cheap to produce. Pharmacological manipulation of phosphodiesterases (PDEs), which hydrolyse cyclic Adenosine Monophosphate (cAMP), have been extensively studied in humans and found to have great therapeutic effect. Kinetoplastid genomes code for the same set of cyclic nucleotide-specific class 1-type phosphodiesterases, with catalytic domains similar to those of human PDEs. The locus of Trypanosoma brucei PDEB1/2 was found to be essential, by either genetic manipulation or the use of the pharmacological inhibitor CpdA, but therapeutic exploitation of TbPDEB1 has so far been hampered by its catalytic domain similarity to human PDEs. However, investigating the unique downstream cAMP signalling cascade, which includes the recently identified cAMP Response Proteins (CARPs), could reveal potentially new trypanosome-specific therapeutic targets. In this study we show that single knockout (sKO) of the CARP genes causes a decreased susceptibility to CpdA, and that null mutants of CARP2-4 display significantly increased intracellular and extracellular cAMP levels. A double knockout (dKO) of CARP2 also shows a significant growth defect. Conversely, overexpression of CARP3 causes a growth delay in both WT s427 and CpdA resistant (R0.8) cells, when exposed to CpdA, and were more sensitive to CpdA compared to other CARP overexpressing cells as well as the WT s427 and R0.8 controls. These cells also have significantly higher intracellular and extracellular cAMP levels relative to the control lines and the other CARP overexpressors. In the cells overexpressing CARP3, whether WT s427 or R0.8, the cellular content of both CARP3 messenger Ribonucleic Acid (mRNA) and protein decreases extensively within 6 h of CpdA exposure. The CARP3 protein has domains that are indicative of a role in protein-protein interaction, signalling, regulation and degradation and probably undergo acylation. Some experimental confirmation of these traits was obtained, using Co-immunoprecipitation (Co-IP) and Mass Spectrometry (MS), with the identification of Adenylyl cyclase (AC) GRESAG4s (also found through RNAinterference Target Sequencing (RITseq) and confirmed by quantitative Reverse Transcription PCR), and proteasome regulatory proteins (PRNs) in addition to membrane and flagellar binding proteins, as potential interactors. Preliminary Immunofluorescence (IF) microscopy showed that CARP3 localizes to plasma membrane ad the flagellum, CARP2 to specific bodies/organelles in the cytosol and CARP1 in the cytosol. RNA sequencing of overexpressing CARP3 reveals differentially expressed proteins involved in cell cycle and cytokinesis as well as transport proteins with several transmembrane domains, consistent with the proposed acylated membrane localisation, and with interaction of CARP3 with membrane proteins and ACs (GRESAG4 isoforms).Thus CARP3 has the domains, interactions and localization consistent with a regulatory role in cAMP metabolism. Thus the CARPs and especially CARP3 are interesting biological molecules providing key new insights into signalling and the cell biology of the trypanosome.

Adenosine is precursor and a metabolic intermediate of adenosine triphosphate in energy transfer, and cyclic adenosine monophosphate in signal transduction. Recent studies have demonstrated that the role of adenosine in the body is much more than just structural as it can also behave as an important regulator of homeostatic functions. Adenosine signalling relies on the activation of the A₁, A₂A, A₂B and A₃ adenosine receptors. Through the development of pharmacological tools and genetic knockout mouse models of specific receptor subtypes, the involvement of these receptors in various physiological systems is quickly being established. This thesis investigates the function of adenosine in the digestive system and specifically how adenosine regulates the release of gastric and pancreatic peptides. With the use of a novel vascularly perfused isolated mouse stomach model and specific A₁ and A₂A receptor knockout animals, the role of adenosine on the release of somatostatin and ghrelin was determined. Lower concentrations of adenosine can inhibit the release of somatostatin and ghrelin via the activation of A1 receptors, while higher concentrations can stimulate their release via activation of A₂A receptors. Given the importance of somatostatin in regulating gastric acid secretion and motility, and ghrelin in regulating systemic energy balance, better understanding of how the release of these two peptides is regulated may reveal potential therapies for eating disorders, gastrointestinal dysfunctions and metabolic diseases. In the pancreas, adenosine was shown to regulate both insulin and glucagon secretion from the pancreatic islets. Studies presented in this thesis demonstrate that adenosine signalling interacts with the effects of the incretin hormone GLP-1 in the pancreas such that concomitant administration of adenosine and GLP-1 in the perfused pancreas induced greater insulin release than GLP-1 administration alone. Furthermore, A₁ receptor knockout mice exhibited more frequent pulses of insulin secretion, which may have contributed to their superior glucose tolerance compared to wild type control mice. These findings on the role of adenosine signalling in the pancreas may have implications in the etiology of diabetes mellitus. The involvement of adenosine signalling in the digestive tract further illustrates the importance of adenosine as a metabolic regulator in homeostasis.

Platelet activation is a critical physiological event, whose main role is to prevent excessive blood loss and repair vessel wall injuries. However, platelet activation must be controlled to prevent unwanted and exaggerated responses leading to the occlusion of the blood vessel. The endothelial-derived inhibitors prostacyclin (PGI2) and nitric oxide (NO) are known to play a critical role in the control of platelet activity, although the mechanism underlying their actions remains unclear beyond the triggering of cyclic nucleotides signaling pathways. The aim of this study was to improve our understanding of platelet regulation by cAMP signaling networks. We observed differences in cAMP signaling depending on the agonists used. Using phosphorylation of PKA substrates as a marker of PKA activity, it was observed that PKA substrates were phosphorylated and dephosphorylated at different time points in a unique temporal pattern. Consistent with this observation we found that individual PKA isoforms, PKA I and II, were localized in distinct subcellular compartments, with PKA I being identified as a lipid raft protein. Our experimental data suggest that the localization of PKA I to lipid rafts is mediated by interaction with A-kinase anchoring proteins (AKAPs). Additionally, PKA signaling events were reversed when potential PKA type I interactions with AKAPs were disrupted with competitive peptides. Using this approach we found that the redistribution of PKA I to lipid rafts facilitated the phosphorylation of GPIbβ and the inhibition of von-Willebrand factor-mediated aggregation. Our data also demonstrated for the first time that the chemical disruption of lipid rafts increased platelet sensitivity to PGI₂, through increased cAMP production and PKA activity. The mechanism by which this occurs may involve sequestering a population of adenylyl cyclase 5/6 to a location remote from Gαs. In conclusion, data presented in this thesis suggest differential roles of PKA subtypes in the regulation of platelet activity. This involves, at least in part, the localisation of PKA I into specific subcellular compartments through an interaction with AKAPs. The potential presence of PKAII-AKAP interactions and the identification of specific AKAPs will be the main aim of future work.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). One of the pathological hallmarks of MS is the T cells-mediated destruction of myelin sheath, which result in axonal damage and subsequent neurological dysfunction. Current MS therapies are focused on immunosuppression as they are aimed at limiting the entry of immune cells into CNS, thereby preventing neuroinflammation. Although these therapies have been shown to be potent disease-modifying agents they fail to prevent or reverse disease progression. Astrocytes, among CNS resident cells, has been recently suggested as alternative highly promising therapeutic targets because of the key role played by these cells in driving disease progression in MS. Indeed, thanks to a close contact between astrocyte end-feet and blood vessels, the crosstalk of astrocytes with encephalitogenic T cells is centrally implicated in MS pathogenesis (Ponath et al., 2018). In this view, we have recently demonstrated that ShcC/Rai is as a novel astrocytic adaptor whose loss in mice accounts for a reduced demyelination and a milder experimental autoimmune encephalomyelitis (EAE), notwithstanding a higher frequency and pathogenicity of autoreactive T cells infiltrated within CNS highlighting the key role played by astrocytes in T cell modulation during EAE (Ulivieri et al., 2016). In the first part of this project we have investigated the molecular mechanism underlying the ability of ShcC/Rai-deficient astrocytes to generate an efficient T cell suppressive microenvironment in the pathological setting of EAE. At the beginning, we focused to study the ability of astrocytes to control the balance between extracellular ATP and adenosine in response to encephalitogenic T cells. We found that astrocytes respond to autoreactive T cells injury by enhancing the expression and activity of CD39 ectonucleotidase, responsible for the enzymatic hydrolysis of extracellular ATP into the immunosuppressive mediator adenosine, and that ShcC/Rai couples CD39 to its negative regulator RanBPM thereby limiting its activity. Accordingly, we measured high adenosine concentration in conditioned medium of Rai-/- astrocytes. As a result, T cells in the presence of microenvironment shaped by Rai knock-out astrocytes showed reduced proliferation and an up-regulation of inhibitory receptor CTLA-4, indicating that higher levels of adenosine are responsible to immunosuppression. We further characterized the impact of Rai on the protein composition of astrocytes-derived extracellular vesicles (EVs) in response to T cell-derived cytokines. Data obtained using a proteomic approach revealed that several proteins are differentially present in EVs released from Rai deficient or control astrocytes. Interestingly, enrichment analysis showed that these proteins participate in glutamate metabolism, in the control of protein folding and in the protection from oxidative stress suggesting that Rai controls pathways involved in brain homeostasis. Additionally, we examined functional polarization of astrocytes towards a neuroprotective (A2) or a neurotoxic (A1) phenotype. We show that Rai-/- astrocytes skew towards the A2 neuroprotective phenotype in response to encephalitogenic T cells both in vitro and in the EAE mouse model of MS by enhancing the activation of STAT3 transcription factor. In the second part of the project we have analyzed the role of ShcC/Rai in adenosine signaling in T cells. We identify a novel mechanism by which Rai in T cells dampens immunosuppressive effect of adenosine by inhibiting A2A receptor signalling interfering with the activation of transcription factor CREB. Characterization of molecular mechanism in a Jurkat T cell lines overexpressing Rai shows that Rai forms a complex with CREB upon A2AR triggering. In this respect, the phosphorylation/activation of CREB was significantly higher in Rai knock-out T cells compared with control following A2AR stimulation. Collectively, these data identify Rai/ShcC adaptor protein as critical regulator of astrocytes responses to T cells mediated neuroinflammation and highlight a new molecular mechanism to which Rai prevents establishment of an immunosuppressive program in T cells by limiting the transcriptional activity of nuclear factor CREB.

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
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Adenosine is an endogenous and ubiquitous nucleoside that exerts many biological functions through interaction with 4 distinct subtypes of G protein-coupled receptors divided into A1, A2A, A2B, and A3. This nucleoside plays an important role in modulating inflammation and tumorigenesis. In the past most of the anti-inflammatory effects of this nucleoside were thought to be due to the activation of the A2A subtype, however more recently, the involvement of the A3 subtype has been also considered relevant for the outcome of inflammation. The figurative elements of blood are important substrates on which adenosine plays multiple physiological functions. In these reviews we summarized the status of the art on the role of the A3 receptor in different types of immune cells including neutrophils, eosinophils, lymphocytes, monocytes, macrophages and dendritic cells. It is known that the interaction with the adenosine A3 receptor inhibits several activities of these cells including the release of TNF-alpha and other potent inflammatory cytokines such as IL-6 and IL-8 and increases the production of IL-10 with anti-inflammatory activity. Recent studies have shown the presence of A3 receptors on neutrophils, which represent the majority of circulating leukocytes and are the first cells to be recruited into a site of tissue inflammation in defending the body against infection. In particular, the activation of the A3 receptor on this cellular type leads to the inhibition of degranulation and superoxide anion production with consequent anti-inflammatory effects. Finally, in lymphocytes, activation of A3 receptor subtype would result in a reduction of the accession of killer T cells to tumor cells by exerting an immunosuppressive effect and suggesting a role for antagonists of this receptor as anti-tumoral drugs. In particular, it was recognized more than two centuries ago a close relationship between inflammation and tumorigenesis, because the tumors arise more easily where there are foci of inflammation; the inflammatory cells are present in tumors and the overexpression of cytokines and chemokines may lead to the onset of tumors; also similar molecular targets and intracellular pathways are activated or dysregulated inflammation and in tumors. Several studies in literature and obtained in our laboratory have shown that adenosine exerts important modulatory function in the growth of tumors, giving an essential role in this to the A3 receptor. Therefore the aim of this study was to evaluate the involvement of adenosine on the regulation of metalloproteinases and in particular of MMP9 in U87MG glioblastoma cells. The metalloproteinases (MMPs) are a family of enzymes able to degrade and remodel the extracellular matrix. They are involved in many physiological and pathological processes, including inflammation and tumor growth. In particular, the degradation of extracellular matrix (ECM), which exerts a mechanical and biochemical barrier to cell movement, was demonstrated to be an important biological process in the invasion and the tumor metastatic process. In particular, it was demonstrated that MMP9 facilitates in vitro invasion of glioblastoma cells and the up-regulation of this metalloproteinase is associated with the progression of malignant glioma in vivo. The results of this study, obtained by using real time RT-PCR and Western blotting, show that adenosine is able to increase both MMP9 mRNA and protein levels through the activation of the A3 adenosine receptor. We also noted that the A3 receptor stimulation led to increased levels of MMP9 protein in cellular extracts of U87MG cells, through phosphorylation of ERK1 / 2, JNK, Akt / PKB and the transcription factor AP-1. The A3 receptor activation also led to an increase in extracellular levels of MMP9 in the supernatants of glioblastoma cells as evaluated by ELISA and gelatine zymography assays. Finally, as for the physiological relevance of the A3 receptor-mediated stimulation of MMP-9 we found that the A3 agonist was responsible for an increase of the invasive ability of U87MG cells. Overall, these results suggest that adenosine, through activation of the A3 receptor, modulates MMP9 protein levels and plays a role in the invasion of U87MG cells.

L’adénosine extracellulaire appartient à la voie de signalisation purinergique et réguledivers processus physiologiques à travers l’activation de ses récepteurs spécifiques (adora).La disponibilité de cette purine dans l’espace extracellulaire est régulée par plusieurs ectoenzymesassurant sa production ou sa dégradation, mais également par des transporteurs denucléosides permettant son passage à travers la membrane. Chez l’adulte, le rôle del’adénosine est assez bien connu. Cependant, l’implication de cette purine au cours del’embryogenèse reste très peu étudiée. Pourtant, un excès d’adénosine dans les phasesprécoces du développement est létal chez la souris et l’oursin, démontrant l’importance de larégulation des concentrations de cette molécule de signalisation lors de l’embryogénèse. Lebut de ma thèse est de comprendre le rôle de l’adénosine au cours de l’embryogenèse enutilisant l’amphibien xénope. En effet, ce modèle a permis de mettre en évidence in vivol’implication de l’ADP au cours du développement de l’oeil chez les vertébrés. La premièrepartie de ce projet a permis de caractériser les acteurs de la voie de signalisation del’adénosine chez le xénope afin d’établir la première carte comparative de leur profild’expression embryonnaire. Cette partie a également permis de mettre en avant laphosphatase alcaline alpl pour son profil d’expression particulier, dans le rein et la rétine. Laseconde partie s’est focalisée sur l’étude fonctionnelle de cette enzyme. Les expériences deperte de fonction montrent son implication lors de la formation de ces deux tissus
Extracellular adenosine belongs to the purinergic signalling pathway and regulatesvarious physiological processes through activation of specific receptors named adora. Theextracellular concentration of adenosine is regulated by several ecto-enzymes involved eitherin its generation or in its degradation but also by nucleoside transporters enabling its exitoutside or entry inside the cell. In adults, the functions of adenosine are quite well known,however, the its involvement during embryogenesis remains poorly studied. An excess ofadenosine in early phases of development is lethal in mouse and sea urchins, demonstratingthe importance of the extracellular adenosine level regulation during embryogenesis. The aimof my phD is to understand the role of adenosine during embryogenesis using Xenopus as avertebrate model. Indeed, the first in vivo evidence of the implication of the purinergic signallingpathway during vertebrate development, and in particular of ADP during eye formation hasbeen demonstrated using this model. The first part of this project was to characterize all theadenosine signalling pathway actors in Xenopus in order to generate the first comprehensiveand comparative embryonic expression map of these genes. This work allowed me to selectthe alkaline phosphatase alpl for functional studies based on its specific expression profile, inthe retina and kidney. These functional studies, mostly carried out by knockdown experiments,constituted the second part of this phD and showed the implication of this enzyme during theeye and kidney development

Tese de mestrado em Bioquímica (Bioquímica Médica), apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2011
O modelo de sinapse actualmente aceite considera que para o processo de comunicação neuronal contribuem não só os neurónios pré- e pós-sináptico mas também um elemento peri-sináptico, os astrócitos. Estes constituem a população de células mais abundante no cérebro, formando juntamente com a micróglia e os oligodendrócitos a rede de células da glia. Presentemente, sabe-se que os astrócitos não são meros elementos passivos, comunicando activamente com as outras células através do seu mecanismo de excitabilidade que usa iões de cálcio (Ca2+) como mensageiro interno para desencadear a gliotransmissão. Este fenómeno é despoletado pela activação de receptores membranares e consiste na libertação de gliotransmissores (GT) como o glutamato, ATP e D-serina. A libertação de GT parece exercer uma função com características em certa medida semelhantes à libertação de neurotransmissores (NT) pelos neurónios. Esta conversa dinâmica e bidireccional entre neurónios e astrócitos depende da libertação de NT e GT que são reconhecidos por ambos os tipos de células. Desta forma, a libertação de NT do terminal pré-sináptico afecta o terminal pós-sináptico mas também os astrócitos envolventes, induzindo um aumento nos seus níveis intracelulares de Ca2+ [Ca2+]i. Em resposta, os astrócitos libertam GT, por um mecanismo não complemente elucidado, afectando novamente o terminal pós-sináptico e ainda a libertação de NT do terminal pré-sináptico. Através destes mecanismos, os astrócitos modulam a excitabilidade neuronal e a transmissão sináptica, havendo evidências de que também modulam fenómenos como a potenciação de longa duração que está intimamente ligada à memória e aprendizagem. As purinas, nomeadamente o ATP e a adenosina, modulam a gliotransmissão, regulando a transmissão sináptica e a comunicação neurónio-astrócito. O ATP desencadeia a sinalização por Ca2+, sendo um dos agentes dos primeiros eventos no processo de gliotransmissão em astrócitos. O aumento da [Ca2+]i mediado pelo ATP deve-se à activação de receptores metabotrópicos (P2YR), que levam à libertação de Ca2+ dos compartimentos intracelulares via fosfolipase C (PLC) e inositol (1,4,5)-trifosfato (IP3), e de receptores ionotrópicos (P2XR), que medeiam a entrada directa de Ca2+ do exterior da célula. O ATP é convertido extracelularmente em adenosina e ambas as purinas se acumulam simultânea ou sequencialmente, estando os seus níveis aumentados em situações patológicas. Os receptores para o ATP (metabotrópicos P2YR e ionotrópicos P2XR) e para a adenosina (metabotrópicos A1R, A2AR, A2BR e A3R) são coexpressos nos astrócitos, o que sugere uma possível interacção entre estas duas famílias de receptores. Da família dos receptores de ATP, o P2Y1R exerce um papel preponderante no desencadear da gliotransmissão. Sabe-se que a adenosina modula a sinalização de Ca2+ desencadeada por neurotransmissores e neuromoduladores como o glutamato e ATP, no entanto, não se compreende completamente quais os receptores de adenosina envolvidos neste processo. É de salientar ainda que os receptores A1 estão negativamente acoplados ao AMP cíclico (cAMP) através de uma proteína Gi inibitória, diminuindo a libertação de GT. Os receptores A2A estão positivamente acoplados ao cAMP através de uma proteína Gs estimuladora, potenciando a libertação de GT. Estudos anteriores mostram que a adenosina modula diferencialmente a internalização de GABA em astrócitos corticais através dos seus receptores membranares de alta afinidade A1 e A2A. É de salientar que esta modulação depende da cooperação entre os receptores A1 e A2A. O principal objectivo deste trabalho é avaliar o papel dos receptores de adenosina, em particular do A2A, na modulação da sinalização por Ca2+. Pretende-se também explorar a possível cooperação entre os receptores A1 e A2A de adenosina no efeito modulatório. As respostas de Ca2+ em astrócitos corticais primários foram estudas pela técnica de imagiologia de Ca2+, usando um microscópio invertido de epifluorescência. O fluoróforo escolhido foi o Fura-2AM que é altamente sensível ao Ca2+, ligando este ião de forma estável e reversível. Trata-se de um fluorocromo raciométrico, uma vez, que sofre um desvio no comprimento de onda do pico de absorvência quando ligado ao Ca2+, de 380 (F380) para 340 (F340) nm. O rácio F340/F380 é usado para avaliar as variações da [Ca2+]i. A internalização do fluoróforo processa-se durante 45 minutos a 37oC e ocorre de forma passiva devido aos grupos AM que atribuem carga global negativa. Já dentro da célula, os grupos AM são clivados por enzimas e o fluoróforo fica retido no interior da célula. As células foram local e brevemente (0,2 segundos) estimuladas com 10 μM ATP, a 22 oC, e a amplitude das respostas foi medida na presença de diferentes fármacos. Foi usado um análogo estável da adenosina, 2-cloroadenosina (CADO), um agonista e um antagonista selectivos para o receptor A2A, CGS 21680 e SCH 58261, respectivamente, e um antagonista selectivo para o receptor A1, DPCPX. No protocolo desenvolvido e optimizado no decurso do projecto, as células foram estimuladas de 5 em 5 minutos e obteve-se respostas para a situação controlo, apenas com tampão HEPES no meio, de seguida na presença do fármaco, que é mantido no meio de perfusão no mínimo durante 15-20 minutos, seguindo-se finalmente a lavagem com tampão HEPES ou com uma mistura de agonista/antagonista do receptor. Como controlo interno, foi ainda obtida a resposta máxima da célula a um estímulo supramáximo, 100 μM ATP. O desenvolvimento deste protocolo dependeu da optimização de uma serie de parâmetros como o período de intervalo entre estimulações, a concentração de ATP usada como estimulo, a duração de cada estimulação, a temperatura da incubação com o fluoróforo e a temperatura de perfusão usada durante toda o registo de sinais de Ca2+. As culturas primárias foram caracterizadas quanto às populações de células presentes, astrócitos, neurónios e micróglia, pela técnica de imunocitoquímica, recorrendo a anticorpos que marcam especificamente estas células. Detectou-se ainda a presença dos receptores de ATP, P2Y1, e de adenosina, A1 e A2A, pela técnica de western blot, aplicando anticorpos específicos para estas proteínas. Observei que a estimulação breve com 10 μM ATP (0,2 segundos) induz um transiente de Ca2+ rápido. A estimulação prolongada (≥ 20 segundos) com ATP induz uma resposta de Ca2+ bifásica, constituída por um pico inicial, seguindo-se um patamar na resposta. Segundo outros autores, o pico inicial é mediado por receptores P2Y e o patamar por receptores P2X. O envolvimento dos receptores P2Y no pico inicial foi comprovado na presença de baixa concentração de Ca2+ extracelular. O análogo estável da adenosina que apresenta afinidade semelhante para os receptores A1 e A2A, 1 μM CADO, potenciou marcadamente a resposta de Ca2+ desencadeada pelo ATP (aumento da resposta: 3,19 ± 0,30 vezes, n=72 células). O agonista selectivo para o receptor A2A, 30nM CGS 21680, imitou este efeito (2,92 ± 0,32 vezes, n=80 células). Estes efeitos foram completamente revertidos após remoção do agonista do meio de perfusão. O parâmetro ‘aumento da resposta’ é o rácio entre a amplitude da resposta ao ATP na presença do fármaco e a amplitude da resposta na ausência de fármaco. Foi ainda calculado o parâmetro ‘resposta controlo/resposta máxima’, tendo observado uma relação inversa entre os valores de aumento de resposta e resposta controlo/resposta máxima. Desta forma, as células que apresentam uma menor resposta controlo em relação à resposta máxima, determinada na presença de 100 μM ATP, sofrem um aumento mais elevado induzido pelos agonistas, CADO e CGS 21680. Tal variabilidade de respostas mostra que estamos perante uma população de células heterogéneas. Para estudar a possível cooperação e/ou interacção entre os receptores A1 e A2A, foram usados antagonistas específicos para estes receptores de forma a testar se revertiam a potenciação induzida pelos agonistas. Verifiquei que os antagonistas, 50 nM SCH 58261 e 50 nM DPCPX, por si só, não induziam variação apreciável na resposta de Ca2+. Surpreendentemente, a potenciação induzida pelo CADO (agonista não-selectivo A1 e A2A) e pelo CGS 21680 (agonista selectivo A2A) foi completamente revertida não só pelo antagonista específico do receptor A2A, SCH 58261, mas também pelo antagonista específico do receptor A1, DPCPX. Tais resultados mostram que a activação de ambos os receptores, A1 e A2A, é fundamental para que o efeito modulatório se verifique, sugerindo a cooperação entre estes receptores. A caracterização das culturas primárias pela técnica de imunocitoquímica mostrou que estas são constituídas maioritariamente por astrócitos (> 90%) e alguma micróglia. Desta forma, pode-se afirmar que estamos perante culturas primárias enriquecidas em astrócitos. A introdução de um passo extra de agitação ao dia 6 de cultura, antes de replaquear as células, permite obter culturas com uma maior percentagem de astrócitos (> 95%) em relação à micróglia. Recorrendo à técnica de western blot foi possível detectar a presença dos receptores de ATP, P2Y1, e de adenosina, A1 na cultura enriquecida em astrócitos. No entanto, usando esta técnica, não foi possível detectar os receptores A2A de adenosina. Uma vez, que a presença de receptores A2A já foi observada anteriormente em astrócitos corticais e que pela técnica de imagiologia de Ca2+, aqui aplicada, foram obtidas respostas a fármacos específicos para estes receptores, pode-se afirmar que estes receptores estão presentes nas culturas primárias usadas neste estudo. Provavelmente a sua densidade será baixa e desta forma difícil de quantificar por western blot. Os resultados aqui apresentados permitem concluir que a adenosina, através da activação de receptores A2A, facilita a sinalização por Ca2+ induzida pelo ATP, em astrócitos corticais. Mais ainda, a cooperação entre os receptores A1 e A2A, parece ser essencial para a modulação observada. O envolvimento de receptores A2A na modulação da sinalização de Ca2+ aqui demonstrado contrapõe resultados anteriores nos quais outros autores mostraram que estes receptores não tinham qualquer efeito sobre a modulação das respostas de Ca2+. Esses autores mostraram ainda que os receptores A2B de adenosina medeiam a modulação da sinalização por Ca2+. No entanto, no trabalho anterior foram usadas concentrações de fármacos não selectivas para os receptores correspondentes, o que poderá justificar as diferenças nos resultados obtidos. Os resultados aqui apresentados estão de acordo com o modelo tetramérico - A1-A1-A2A-A2A - de receptores de adenosina proposto para a modulação diferencial da internalização de GABA pela adenosina, em astrócitos corticais. Os resultados aqui obtidos apoiam um cenário semelhante para a modulação da sinalização de Ca2+ pela adenosina com possíveis implicações na libertação de GT. Trata-se por isso de um novo mecanismo de modulação sináptica indirecto que é provavelmente mediado pela cooperação dos receptores A1 e A2A de adenosina, permitindo uma regulação apertada e uma transição suave entre os estados inibitório e potenciador.
Purines, namely ATP and adenosine, modulate gliotransmission, regulating synaptic transmission and ultimately neuron-astrocyte communication. ATP triggers the signalling of free calcium ions (Ca2+), which is the substrate for gliotransmission, in astrocytes. ATP is extracellularly converted into adenosine and both purines can be accumulated simultaneously or sequentially, acting on a plethora of membrane astrocytic receptors. Adenosine modulates GABA uptake in cortical astrocytes through A1 (A1R) and A2A (A2AR) receptors. Most importantly, this modulation is dependent on tight A1R-A2AR cooperation. Therefore, the aim of this project was to evaluate the role of adenosine receptors, mainly A2AR, in Ca2+ signalling modulation and also to explore the possible cross-talk between A1R and A2AR in this modulation. Ca2+ responses of cortical primary astrocytes were studied by Ca2+ imaging technique using Fura-2AM, at 22oC. Cells were locally and briefly (0.2 seconds) stimulated with 10μM ATP and the amplitude of fluorescence signals was measured in the presence of different drugs. Cultures were further characterized by immunocytochemistry and western blot techniques. A stable adenosine analogue, 2-Chloroadenosine (1 μM), markedly enhanced ATP-induced Ca2+ responses (fold increase: 3.19 ± 0.30, n=72 cells). An A2AR selective agonist, CGS 21680 (30nM), mimicked this effect (2.92 ± 0.32, n=80 cells). The potentiation mediated by both agonists was not only completely reverted by an A2AR selective antagonist, SCH 58261 (50nM), but also by an A1R selective antagonist, DPCPX (50nM). Primary enriched astrocytic cultures are constituted mainly by astrocytes (> 90%) and microglial cells. The presence of ATP, P2Y1R, and adenosine, A1R, but not A2AR could be demonstrated for primary astrocytic cultures. Nevertheless, selective agonists and antagonist used in Ca2+ imaging have proven the presence of functional A2AR. It is concluded that adenosine, through A2AR activation, potentiates ATP-induced Ca2+ signalling in astrocytes. Furthermore, A1R-A2AR cooperation seems to be required for this modulation.

Adenosine is an endogenous nucleoside that has been recognised to be a molecule with autocrine/paracrine functions, acting as a signal molecule to preserve host defence and tissue integrity during inflammation and trauma in addition to its important role as homeostatic regulator. The physiological activities of adenosine involve its interactions with four types of receptors, designed as A1, A2A, A2B and A3. Adenosine mediates its anti-inflammatory activity primarily through the A2A receptor (A2AAR). The ecto-5’nucleotidase/CD73 degrades AMP to adenosine and represents a key enzyme for adenosine accumulation at the site of injury. The aim of this research work was to explore different aspects of adenosine signalling pathway in inflammation. Several findings implicate the adenosine signalling pathway as an innate mechanism to attenuate excessive tissue damage and identify CD73 as critical control points for endogenous adenosine generation. It has been shown that CD73 plays an important role in regulating vascular permeability and leukocyte trafficking in inflammatory disease; and a crucial role in the regulation of immune/inflammatory cell function. A better understanding of the role of CD73 enzyme in the development of inflammatory processes can help to identify new therapeutic strategies aimed at strengthening the endogenous anti-inflammatory mechanisms. For this reason we sought to investigate on the role of CD73, the key enzyme in “switching on” adenosine signalling, in the development of inflammation through its pharmacological blockade by using the selective inhibitor, adenosine 5'-(α,β-methylene) diphosphate (APCP; 400 μg/site), in an in vivo model of acute inflammation represented by carrageenan-induced pleurisy in rats. We found that local inhibition of CD73 significantly increased cell accumulation, exudate formation and pro-inflammatory cytokine content into the pleural cavity in the acute phase of inflammation with no differences in the sub-acute phase. The in vivo treatment with APCP induced cells recruited into the pleural cavity to change in a phenotype with increased ability to migrate in vitro either in the presence or in the absence of a chemotactic stimulus. In parallel, these cells showed a reduced CD73 expression and activity compared to cells collected from control group. In addition, APCP, in vitro, strongly increased the ability of cells from control groups to migrate in the presence of a chemotactic stimulus. Local inhibition of CD73 increased also the infiltration of the lung with polymorphonuclear leukocytes (PMNs) and the degree of lung injury 4 hours following carrageenan injection. The interest to explore the role of adenosine signalling pathway in the control of inflammation has been growing further following evidence that adenosine signalling is also involved in the mechanism of action of some well-known anti-inflammatory drugs. With regard to this, we focused our interest on the possible involvement of adenosine signalling in the anti-inflammatory mechanism of nimesulide, in vivo (rat paw oedema) and in vitro (J774A.1cell line); indeed, there is evidence that nimesulide anti-inflammatory effect is the consequence of regulation of the production and actions of a wide range of inflammatory mediators, independently from the sole cyclooxygenase-2 (COX-2) enzyme inhibition. To date, the molecular mechanisms at the basis of nimesulide peculiar pharmacological effects are still unclear. In vivo, in the model of carrageenan-induced rat paw oedema, we found that the anti-inflammatory effect of nimesulide (5 mg/kg i.p.) was inhibited by pre-treatment with the adenosine A2A receptor antagonist, ZM 241385 (3 mg/kg i.p.), and by local administration of the CD73 inhibitor, APCP (400 μg/paw). Furthermore, we observed increased activity of 5'-nucleotidase/CD73 in plasma and paws of nimesulide-treated rats, 4 h following oedema induction that represented the inflammatory peaking point. In vitro, the inhibitory effect of nimesulide on nitrite and prostaglandin (PG)E2 (PGE2) production by lipopolysaccharide (LPS)-activated J774 macrophage cell line was again reverted by ZM 241385 and APCP. Furthermore, nimesulide increased CD73 activity in J774 macrophages while it did not inhibit nitrite accumulation by LPS-activated small interfering RNA (SiRNA) CD73 silenced J774 macrophages. Our data demonstrate that the anti-inflammatory effect of nimesulide is, in part, mediated by CD73-derived adenosine acting on A2A receptors. There is evidence that A2AAR activation beside anti-inflammatory effects promotes wound healing and extracellular matrix production; given that extracellular matrix and fibroblasts take an active part in the modulation of inflammation beside wound healing, we investigated whether and how extracellular matrix was involved in the anti-inflammatory effect of A2A receptor. Specifically, we evaluated changes in tissue fibroblast growth factor-2 (FGF-2), an important growth factor for fibroblasts that has been shown to facilitate not only tissue regeneration but also to dampen inflammation, following systemic administration of the A2A agonist, CGS 21680, in a rat model of acute inflammation (paw oedema). We observed that CGS 21680 prevented oedema development and inflammation, confirming an anti-inflammatory effect of A2AR. The effect of CGS 21680 was specific, through A2A adenosine receptor stimulation, as revealed by co-administration with ZM 241385 that reverted CGS 21680 inhibitory effect. On the basis of histological analysis showing an increased matrix deposition following rat treatment with CGS 21680, we evaluated whether the beneficial effect of A2A agonist, CGS 21680, was paralleled by changes in FGF-2 expression. Interestingly, we found that the expression of FGF-2 in rat paws, evaluated at each hour following carrageenan injection, was increased following rat treatment with CGS 21680. Immunofluorescence analysis confirmed data obtained by western blot and also showed spots of co-localization between A2AR and FGF-2. In conclusion, in this research work we demonstrate that CD73 regulates cell migration in the acute phase of inflammation and that the anti-inflammatory effects mediated by A2AR activation are paralleled by changes in extracellular matrix morphology. These findings suggest the important role of CD73/adenosine/A2A signalling in the control of the acute phase of inflammation, characterised by PMNs infiltration, but also in the control of a late phase, characterised by re-arrangement of extracellular matrix. In addition, we also demonstrate that CD73/adenosine/A2A axis is involved in the mode of action of nimesulide. Our study may open a path to re-evaluate the mechanism of action of nimesulide and to identify new therapeutic opportunities in COX-2 inhibitors which display a more potent activity on adenosine signalling. Furthermore, these results may give the cue to project an innovative anti-inflammatory strategy based on the manipulation of endogenous anti-inflammatory pathways.

Tese de mestrado em Biologia Molecular e Genética, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, em 2018
Os astrócitos são células multifuncionais que coexistem com os neurónios no sistema nervoso central, cuja morfologia pode variar conforme localização e/ou função. Na sinapse, interagem direta e indiretamente com os terminais pré-sinápticos, pós-sinápticos, e astrócitos vizinhos, uma vez que possuem recetores específicos para neurotransmissores, sendo capazes de libertar gliotransmissores que, por sua vez, se ligam a recetores presentes nos terminais nervosos. Esta comunicação bidirecional entre astrócitos e neurónios leva então ao conceito de sinapse tripartida. Além da gliotransmissão, os astrócitos também são responsáveis pela maioria da captação de glutamato nas sinapses. O glutamato é o principal neurotransmissor excitatório com um papel crucial na sinalização neuronal. Após ter cumprido a sua função como neurotransmissor, é necessário a saída rápida deste da fenda sináptica de modo a impedir excitotoxicidade. Para tal, astrócitos e neurónios expressam transportadores de glutamato. A atividade destes transportadores é controlada de forma fina por vários componentes intra e extracelulares como fatores de crescimento, neuromoduladores, sinalização de cálcio, etc. Atualmente, cannabis é a droga ilícita mais consumida nos países desenvolvidos. A planta cannabis sativa possui centenas de substâncias psicoativas, como o THC, que se ligam a recetores específicos no cérebro e periferia que, iniciando uma cascada de eventos, levam aos efeitos psicoativos da cannabis, já conhecidos. Hoje em dia sabe-se que os mamíferos são capazes de produzir e degradar endocanabinóides que também se ligam a estes recetores funcionando como neurotransmissores retrógrados. O sistema endocanabinóide passou a ser conhecido por um sistema regulador homeostático com uma sinalização intra e intercelular. Os recetores de canabinóides mais estudados são os recetores de cannabinóides tipo 1 (CB1R) e recetores de cannabinóides tipo 2 (CB2R). O CB1R tem uma expressão mais dominante no sistema nervoso central (SNC) enquanto o recetor CB2 é mais expresso no sistema imunitário. Durante a transmissão sináptica, os terminais pré-sinápticos libertam neurotransmissores na fenda sináptica que, por sua vez, se vão ligar aos recetores expressos nos terminais pós-sinápticos. Uma vez ligados, dependendo do tipo de neurónio/célula, os neurotransmissores podem conduzir a uma variedade de respostas, como inibição, excitação e/ou síntese de segundos mensageiros. Por vezes, os terminais pós-sinápticos respondem ao estímulo através da síntese de endocanabinóides. Estes, por sua vez, são libertados na fenda sináptica ligando-se e ativando os recetores canabinóides. No cérebro, a atividade desses recetores pode afetar múltiplas funções biológicas, como perceção da dor, digestão, aprendizagem, memória, ansiedade e funções cognitivas. Isso é feito através da regulação da transmissão sináptica e da plasticidade, como a inibição da libertação do transmissor, o controlo da excitabilidade neuronal e a regulação da plasticidade sináptica de curto e longo prazo. O CB1R é um recetor acoplado à proteína G (GPCR) do sistema canabinóide com sete domínios transmembranares. A expressão deste não é uniforme ao longo do cérebro, sendo mais expresso no cerebelo, gânglios basais e córtex. Todavia, a baixa expressão do recetor não significa necessariamente que a sua função não é relevante. Nos neurónios, os CB1Rs são geralmente acoplados a Gi/o e, em alguns casos, a Gs, enquanto que nos astrócitos há evidências deste recetor estar acoplado a Gq/11. Nestas células, sabe-se que a ativação deste recetor leva à mobilização de cálcio das reservas intracelulares através da atividade de fosfolipase C (PLC). Semelhante ao CB1R, os recetores de adenosina são um grupo de proteínas GPCR que são ativadas por ligação com a adenosina. Até à data, quatro subtipos de recetores de adenosina foram descobertos e clonados: A1, A2A, A2B e A3. Os recetores A1 e A3 de adenosina são inibitórios sendo acoplados a Gi/o inibindo a acumulação de cAMP e a atividade de proteína quinase A (PKA), enquanto que os receptores A2A e A2B são excitatórios, acoplados a proteínas Gs cuja ativação leva ao aumento de cAMP e ativação de PKA. No SNC, a adenosina é considerada um neuromodulador potente e os recetores A1 e A2A de adenosina estão associados a funções cerebrais críticas, como a libertação e captação de neurotransmissores e plasticidade sináptica. Nos astrócitos, a atividade dos recetores A2A de adenosina está correlacionada com a captação diminuída de aspartato e a regulação da memória, enquanto que os recetores A1 estão correlacionados com a ativação da PLC, sono e fatores de crescimento. O objetivo deste estudo foi entender melhor a sinalização de cálcio mediada pelo CB1R e averiguar a sua relevância na atividade de transportadores de glutamato em astrócitos, assim como estudar a interação entre os recetores de adenosina e o CB1R em astrócitos. Para este fim, foram realizados ensaios de imunocitoquímica, imagiologia de cálcio e captação de glutamato radioativo em culturas primárias de astrócitos. As culturas primárias de astrócitos foram preparadas do córtex cerebral de Sprague Dawley com 0-2 dias, sendo usadas células com 3 semanas em cultura. Nos ensaios de imagiologia de cálcio, foi observada a evolução do cálcio intracelular ao longo do tempo, incubando a cultura de astrócitos com uma sonda FURA-2AM. Nos ensaios de captação de glutamato, foi vista a quantidade de glutamato radioativo incorporada pelos astrócitos. A captação mediada pelo transportador GLAST ou GLT-1 é dada pela diferença entre o transporte total (na ausência de qualquer antagonista) e o transporte obtido quando um destes transportadores é bloqueado. Em imunocitoquímica, a expressão do CB1R, assim como dos principais transportadores de glutamato GLT-1 e GLAST, foi encontrada em culturas primárias de astrócitos. A expressão dos transportadores GLAST e GLT-1 não foi uniforme. O GLT-1 apresentou uma expressão mais alta na área ao redor do núcleo enquanto que o GLAST aparentou ter uma expressão mais alta na membrana e no espaço intracelular. Em imagiologia de cálcio, a presença do agonista seletivo do CB1R induziu transientes de cálcio, sendo que a amplitude e a frequência destes era dependente da concentração de agonista. Na presença de um antagonista seletivo do CB1R, o efeito foi abolido, levando à conclusão que a sinalização de cálcio induzida por ACEA é mediada pela ativação do CB1R. Esta sinalização de cálcio foi abolida na presença de um depletor de reservas de cálcio intracelulares enquanto que, em condição de baixa concentração de cálcio extracelular, a amplitude e frequência diminuíram. Na presença de um antagonista de PLC (U73122), o efeito também foi abolido. Quando a proteína Gi/o foi inibida na presença de um antagonista especifico, PTx, a frequência destes diminuía sem alterações na amplitude, sugerindo fortemente algum tipo de participação por parte de um recetor que não o CB1R acoplado a Gi/o na sinalização de cálcio. Quando o crosstalk entre CB1R e A2AR foi avaliado, observou-se que a pré-ativação de A2AR diminuiu a frequência e a amplitude dos transientes de cálcio mediados por CB1R. Por outro lado, quando o A2AR foi inibido, a frequência de transientes mediada por CB1R aumentou quando comparada à incubação de ACEA isolado. Quando o recetor de adenosina A1 foi previamente inibido, a frequência de transientes induzida pelo CB1R pareceu diminuir. Em ensaios de uptake de glutamato, a presença de ACEA aumentou a captação de glutamato em astrócitos, através de um aumento da constante cinética Vmax na atividade do transportador GLAST sem diferença em Km. Na presença do antagonista do CB1R, este efeito foi abolido, assim como na presença de antagonista de PLC. Já na presença de um antagonista de PKC, uma proteína abaixo na sinalização da via de sinalização de PLC, observou-se uma potenciação do transporte, o que indica que esta proteína é provavelmente não participativa na via. Para saber se a sinalização de cálcio e potenciação do transporte de glutamato mediadas pela ativação do CB1R são efeitos paralelos ou em série, ativou-se o CB1R na presença de um quelante de cálcio. Nesta condição, não se observou potenciação de transporte de glutamato, o que leva a crer que a sinalização de cálcio iniciada pela ativação de CB1R leva à potenciação da atividade do transportador GLAST. Concluindo, em astrócitos, a ativação do CB1R induz transientes de cálcio intracelulares cuja amplitude e frequência têm um comportamento dose-resposta. A ativação de PLC e as reservas de cálcio intracelulares são essenciais para a ocorrência destes transientes. Isto sugere fortemente o acoplamento de Gq/11 ao CB1R, que está de acordo com estudos anteriores. Por sua vez, estes transientes aumentam o transporte de glutamato através do aumento da atividade do transportador GLAST, numa via independente de PKC. A inibição do recetor de adenosina A2A aumenta a sinalização de cálcio mediada pelo CB1R, implicando uma interação direta entre estes dois recetores.
Astrocytes are the major glial cell type in the central nervous system (CNS) being responsible for many actions in the CNS. Astrocytes express several types of G-protein coupled receptors, namely the cannabinoid type one receptors (CB1R) that are activated by endocannabinoids released from neurons. Usually these receptors are coupled to Gi/o proteins although there are reports of Gq/11 coupling. In astrocytes, CB1R activation leads to phospholipase C (PLC)-dependent Ca2+ mobilization from internal cellular stores, which will then have key roles in brain function. Astrocytes also express adenosine A2A receptors (A2AR) that, when activated by adenosine, have several modulatory effects, such as the control of glutamate transport. Thus, both type of receptors, CB1R and A2AR, are of pivotal importance in modulation of neuronal excitability. Interaction between CB1R and A2AR have been well studied in neurons, nevertheless nothing is known concerning this crosstalk in astrocytes. From the several roles of astrocytes in the CNS, the glutamate uptake mediated by specific glutamate transporters is one of the most relevant functions, since this function prevents excitotoxicity due to glutamate accumulation in the synaptic cleft. To achieve the suitable glutamate transport from the extracellular space, astrocytes express mainly the glutamate transporters GLAST and GLT-1. Having in mind the lack of information regarding the effect of CB1R in astrocytes, the main aim of this work was to study the role of CB1R activation-mediated calcium signaling upon GLAST transporter in rat primary astrocytic cultures. This study was designed to better understand the role of CB1R activation on astrocytic calcium signalling and to study its relevance upon glutamate transport. Furthermore, this work aimed to study the crosstalk between CB1R and A2AR upon calcium signaling and glutamate uptake. Primary astrocytic cultures from cortex were prepared from Sprague Dawley pups (0-2 days old). Calcium signaling and [3H]-glutamate uptake experiments were performed with 18-23 DIC astrocytes. Calcium signaling experiments were performed using FURA-2AM dye and specific transport mediated by GLAST was achieved by the difference of total glutamate transport and transport in presence of UCPH-101, a specific blocker of this transporter. In primary cultures of astrocytes it was observed that CB1R activation with ACEA led to calcium transients, whose amplitude and frequency was dose-dependent for the two tested ACEA concentrations (0.5 and 1.0 μM). The occurrence of these transients was abolished in the presence of antagonists of CB1R, AM251 (1 μM), of PLC, U73122 (3 μM) and a chelator of intracellular calcium stores, CPA (10 μM). Low extracellular calcium concentration decreased both frequency and amplitude of the transients while inhibition of Gi/o protein, with PTx (5μg/ml), decreased significantly frequency with no significant changes in amplitude. By performing glutamate uptake assays, it was observed that GLAST was the main glutamate transporter in primary astrocytic cultures. CB1R activation increased significantly the Vmax kinetic constant of the GLAST transporter. This potentiation involved PLC and calcium signalling, but was PKC independent. When evaluating the crosstalk between CB1R and A2AR, it was observed that activation of A2AR decreased both frequency and amplitude of CB1R-mediated calcium transients. On the other hand, when the A2AR was inhibited, CB1R-mediated transients frequency was higher when compared to ACEA incubation alone. When adenosine A1 receptor was previously inhibited, the CB1R-elicited transients ‘frequency seemed to be decreased. In conclusion, CB1R activation triggers calcium transients in astrocytes by PLC activation, in a dose-dependent manner. This calcium signaling then enhances glutamate transport probably by increasing the number of functional GLAST transporters in the plasma membrane. In parallel, adenosine A2AR activation decreases CB1R activity. This highly suggests that, in a synapse context, endocannabinoids released by post-synaptic neurons will activate astrocytic CB1Rs, increasing glutamate transport and contributing to clear up excessive glutamate at the synaptic cleft, and that this effect is possibly controlled by adenosine A2A receptors.

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2014
Brain-derived neurotrophic factor (BDNF) and its high-affinity full-length receptor, TrkB-FL, play a central role in the nervous system by providing trophic support to neurons and by regulating synaptic transmission and plasticity. BDNF signalling is impaired in Alzheimer’s disease (AD), a neurodegenerative disorder characterized, among other features, by the accumulation of the amyloid-β (Aβ) peptide. Although the mechanisms implicated in the reduction of BDNF signalling in AD were not clarified, the reestablishment of BDNF actions is considered as a promising strategy for AD treatment. In last decade it became clear that most of synaptic actions of BDNF, including the ones upon synaptic transmission, plasticity or upon neurotransmitter release, are fully dependent on adenosine A2A receptors (A2AR) activation. However, evidences indicate that A2AR antagonists can prevent the deficits in AD animal models. Given the lack of data clarifying the mechanisms behind the changes on BDNF signalling, namely changes on TrkB receptors, and the knowledge that A2AR activation facilitates most of BDNF synaptic actions, the main goal of this project was to study the impact of Aβ peptides and A2AR on BDNF signalling. This work revealed that in rat primary neuronal cultures Aβ selectively increases mRNA levels for the truncated TrkB-T1 and TrkB-T2 isoforms without affecting TrkB fulllength (TrkB-FL) mRNA levels. Moreover, Aβ increases protein levels of total pool of truncated TrkB receptors (TrkB-Tc) and decreases TrkB-FL protein levels. This effect is explained by the Aβ-induced calpain-mediated cleavage on TrkB-FL receptors, downstream of Shc binding site, which results in the formation of a new truncated TrkB receptor (TrkB-T’) and a new intracellular fragment (TrkB-ICD), which is also detected in post-mortem human brain samples. In hippocampal slices it was observed that Aβ impairs BDNF function in a calpaindependent way, upon modulation of GABA and glutamate release from hippocampal nerve terminals, and upon modulation of long-term potentiation (LTP). Finally, the exogenous BDNF strongly reduces the Aβ-induced activation of caspase-3 and calpain in neuronal cultures, an effect not affected by A2AR agonist or antagonist. Moreover, for the first time it was shown that chronic in vivo blockade of A2AR by a selective antagonist, prevents the facilitatory action of BDNF upon ex-vivo CA1 hippocampal LTP and decreases both mRNA and protein levels of the TrkB-FL receptor in rat hippocampus. In conclusion, the present work shows that Aβ induces a TrkB-FL cleavage mediated by calpain and impairs BDNF-mediated effects in synaptic plasticity and neurotransmitter release in a calpain-dependent way. While the BDNF action upon synaptic plasticity is abolished under chronic in vivo A2AR blocking conditions, the protective actions of this neurotrophin against Aβ toxicity were found to be dependent on A2AR activation.
O factor neurotrófico derivado do cérebro (Brain-derived neurotrophic factor- BDNF) e o seu receptor de alta afinidade, TrkB-FL, desempenham um papel central no sistema nervoso, dado que promovem suporte trófico aos neurónios e que regulam a transmissão e plasticidade sinápticas. A sinalização mediada pelo BDNF encontra-se diminuída na doença de Alzheimer (Alzheimer’s disease -AD), uma doença neurodegenerativa na qual ocorre acumulação do péptido beta amilóide (amyloid-beta -Aβ). Apesar dos mecanismos envolvidos na redução da sinalização mediada pelo BDNF na AD não serem totalmente conhecidos, o restabelecimento das acções do BDNF tem sido considerado como uma estratégia promissora para a terapêutica desta doença. Na última década tornou-se claro que a maioria das acções sinápticas do BDNF, incluindo as acções na transmissão e plasticidade sinápticas e também na libertação de neurotransmissores, é dependente da activação dos receptores A2A da adenosina (A2AR). Contudo, o uso de antagonistas dos A2AR tem sido apontado como uma possível estratégia terapêutica para o tratamento da AD. Dada a falta de evidências que clarifiquem os mecanismos envolvidos nas alterações da sinalização mediada pelo BDNF e o conhecimento de que a activação dos A2AR facilita a maioria das acções sinápticas do BDNF, o objectivo principal desta tese foi estudar o impacto dos péptidos Aβ e dos A2AR na sinalização mediada pelo BDNF. Este trabalho revelou que, em culturas primárias de neurónios corticais, o Aβ aumenta os níveis de mRNA dos receptores TrkB truncados, TrkB-T1 e TrkB-T2, sem afectar os níveis de mRNA dos receptores TrkB completos, TrkB-FL. Por outro lado, verificou-se que o Aβ aumenta os níveis proteicos do conjunto de receptores TrkB truncados e que diminui os níveis proteicos dos receptores TrkB-FL, por um mecanismo independente da proliferação glial e da activação de caspases. Foi ainda possível concluir que o Aβ induz a clivagem, mediada por calpaínas, dos receptores TrkB-FL, esta clivagem dá-se após o local de ligação da Shc e antes do início do domínio de cinase de tirosina, pelo que origina um novo receptor TrkB truncado (TrkB-T’), contendo o local de ligação à Shc, e um novo fragmento intracelular (TrkBintracellular domain- ICD), contendo a totalidade do domínio da cinase. No entanto, a presença destes fragmentos, não mostrou afectar a fosforilação do receptor TrkB-FL induzida pela exposição ao BDNF. Interessantemente, foi possível detectar o fragmento TrkB-ICD em uma amostra, post-mortem, de cérebro humano. Mostrou-se também que a inibição das calpaínas previne as alterações dos níveis proteicos das isoformas do TrkB, induzidas pelo Aβ, sem afectar as alterações ao nível do mRNA do TrkB. Por outro lado, este trabalho revelou que o BDNF exógeno reduz a activação da caspase-3 e das calpaínas induzida pelo Aβ, de uma forma independentemente dos A2AR. Em fatias de hipocampo de ratos adultos, este trabalho mostrou que o Aβ diminui as acções do BDNF na plasticidade sináptica, nomeadamente na potenciação de longa duração (Long-term potentiation, LTP) na área CA1 do hipocampo, bem como no seu efeito sobre libertação de neurotransmissores (GABA e glutamato) de sinaptosomas. Notavelmente, o inibidor das calpaínas, MDL28170, mostrou restabelecer os efeitos do BDNF, na presença do péptido Aβ, tanto na plasticidade sináptica como na libertação de neurotransmissores. Este trabalho permitiu ainda concluir que o bloqueio crónico dos A2AR, in-vivo, através da administração de um antagonista selectivo (KW-6002), previne o efeito potenciador do BDNF na LTP, registada ex-vivo na área CA1 do hipocampo, e que diminui os níveis de mRNA e de proteína do receptor TrkB-FL, no hipocampo de rato. Em suma, o presente trabalho revelou que o péptido Aβ induz a clivagem dos receptores TrkB-FL, mediada pelas calpaínas, e que bloqueia as acções mediadas pelo BDNF na plasticidade sináptica e na libertação de GABA e glutamato por um mecanismo dependente da actividade das calpaínas. Se por um lado, o efeito do BDNF na plasticidade sináptica é perdido aquando da inibição crónica dos A2AR, o efeito protector desta neurotrofina contra a toxicidade induzida pelo Aβ mostrou-se independente da activação dos A2AR.
Fundação para a Ciência e a Tecnologia (FCT)

Tuberculosis continues to be one of the major causes of morbidity and mortality worldwide. Several mycobacterial species such as M. tuberculosis and M. africanum are responsible for causing this disease in humans. Reports of high cAMP levels in mycobacterial species (as compared to other bacteria such as E. coli) suggested that this second messenger may play an important role in the biology of mycobacteria. Further, it was reported that infection with mycobacteria led to an increase in the cAMP levels within the host macrophage. More recent studies have shown that this cAMP increase may be due to bacterially derived cAMP, hinting at a role for cAMP in mycobacterial pathogenesis. Given this background, the study of cAMP in mycobacteria proves to be an interesting field of research. Signalling through cAMP involves an interaction of this cyclic nucleotide with a cAMP-binding protein. These proteins typically contain a cyclic nucleotide-binding domain (CNB domain) linked to another (effector) domain. The CNB domain is thought to allosterically control the activity of the effector domain, thus mediating cellular responses to altered cAMP levels. For example, in the case of eukaryotic protein kinase A (PKA), binding of cAMP to the CNB domain results in relieving the inhibitory effects of the regulatory subunit on the catalytic subunit. The catalytic subunit then phosphorylates its target substrates, eliciting a variety of cellular responses. This work involves the characterisation of novel cAMP-binding proteins from mycobacteria, in an attempt to better understand cAMP signalling mechanisms in these organisms. The genome of M .tuberculosis H37Rv is predicted to code for ten CNB domain-containing proteins. One of these genes is Rv0998 (KATmt). KATmt was found to contain a GCN5 related N-acetyltransferase (GNAT) domain linked to a CNB domain. KATmt finds orthologues throughout the genus Mycobacterium, thereby suggesting its role in the basic physiology of these organisms. In addition, such a domain fusion is unique to mycobacteria and hence promises to deliver insights into the biology of this medically important genus. Presented here are the biochemical and functional characterisation of KATmt and its orthologue from M. smegmatis, MSMEG_5458 (KATms). Recombinant KATms bound cAMP with high affinity, validating the functionality of its CNB domain. Mutational and analogue-binding studies showed that the biochemical properties of the CNB domain were similar to mammalian protein kinase A and G-like CNB domains. The substrate for the GNAT acetyltransferase domain was identified to be a universal stress protein from M. smegmatis (MSMEG_4207). MSMEG_4207 was acetylated at a single lysine residue (Lys 104) by KATms in vitro. Further, cAMP binding to KATms increased the initial rate of acetylation of MSMEG_4207 by 2.5-fold, suggesting allosteric control of acetyltransferase activity by the CNB domain. To ascertain that KATms acetylated MEMEG_4207 in vivo, an in-frame deletion of the KATms gene was generated in M. smegmatis (ΔKATms). MSMEG_4207 was immunoprecipitated from wild-type M. smegmatis and the ΔKATms strains, followed by mass spectrometric analysis. Acetylated MSMEG_4207 was only present in the wild-type strain, confirming that KATms and MSMEG_4207 is an in vivo enzyme-substrate pair. Key biochemical differences were observed between KATms and KATmt. KATmt had an affinity for cAMP in the micromolar range, close to three log orders lower than that of KATms. In addition, KATmt showed strictly cAMP-dependent acetylation of MSMEG_4207. This demonstrates that orthologous proteins often evolve under varied selective pressures, resulting in divergent properties. Using a combination of bioluminescence resonance energy transfer (BRET) and amide hydrogen/deuterium exchange mass spectrometry (HDXMS), the conformational changes that occur upon cAMP binding to the CNB domain of KATms were monitored. A BRET-based conformation sensor was constructed for KATms by inserting KATms between GFP2 (green fluorescent protein) and Rluc (Renilla luciferase). An increase in BRET upon cAMP binding to the sensor was observed. HDXMS analysis revealed that besides the CNB domain, the only other region that showed conformational changes in KATms upon cAMP-binding was the linker region. To confirm that the linker region was important in propagating the effects of cAMP-binding to the acetyltransferase domain, an additional construct for BRET analysis encompassing the CNB domain and the linker region was generated. The magnitude of the increase in BRET was similar to the full length BRET-based sensor, validating the crucial role of the linker region in propagating cAMP-mediated conformational changes. A ‘PXXP’ motif found in the linker region, showed maximum exchange in HDXMS analysis. Mutation of both these proline residues to alanine in KATms, as well as KATmt, resulted in decoupling of cAMP-binding and allosteric potentiation of acetyltransferase activity. In contrast to the intricate parallel allosteric relays observed in other CNB domain-containing proteins, the CNB domain in KATms functions as a simpler cyclic nucleotide binding-induced switch involving stabilization of the CNB and linker domain alone. Therefore, KATms is an example of a primordial CNB domain where conformational changes are a consequence of binding-induced ordering alone. Using a computational approach, putative substrate proteins of KATmt from M. tuberculosis were identified. The substrate specificity of lysine acetyltransferases is determined loosely by a consensus sequence around the lysine residue which is acetylated. Using this property of protein acetyltransferases, the genome of M. tuberculosis H37Rv was mined for proteins harboring lysine residues in a similar sequence context as seen in MSMEG_4207. In vitro biochemical analysis of some of the predicted substrates helped confirm a subset of enzymes belonging to the fatty acyl CoA synthetase (FadD) class as substrates of KATmt. The acetylation of FadDs by KATmt was cAMP-dependent. In each of the four proteins tested, acetylation was found to occur at a single conserved lysine residue. To confirm that FadDs were acetylated by KATmt in vivo, BCG_1055, the orthologue of KATmt in M. bovis BCG, was deleted using the specialised transduction method. FadD13, one of the FadDs acetylated by KATmt in vitro, was immunoprecipitated from wild-type M. bovis and the ΔBCG_1055 strains using a FadD13-specific polyclonal antibody. Acetylated FadD13 was almost completely absent in ΔBCG_1055 but substantial amounts of acetylated FadD13 were present in the wild-type strain, indicating that FadD13 was indeed an in vivo substrate of KATmt. The functional consequences of acetylation of FadDs were analysed using an in vitro fatty acyl CoA synthetase assay. The activities of FadD2 and FadD13 were inhibited on acetylation with KATmt, while acetylation of FadD5 resulted in the formation of a novel product. Therefore, modification of the highly conserved lysine residue in these enzymes by acetylation led to loss or alteration of their enzymatic activity, suggesting that acetylation may be used as a regulatory mechanism to modulate the activities of some of the FadDs by KATmt in a cAMP-dependent manner. Given the extensive role of FadDs in cell wall biosynthesis and lipid degradation in mycobacteria, it seems possible that post-translational control by KATmt in a cAMP-dependent manner constitutes a novel mechanism utilised by these bacteria to regulate these pathways. This direct regulation of protein lysine acetylation by cAMP appears to be unique to mycobacteria, as orthologues of KATmt are not found outside this genus. In addition, the biochemical differences between KATmt and its orthologue from M. smegmatis KATms, indicate species specific variation, on a common theme. This study is the first report of protein lysine acetylation in mycobacteria. In addition to the identification of several proteins subject to this post-translational modification, the effect of acetylation on the enzymatic activities of some of them has been elucidated.

Tese de Doutoramento em Biologia Experimental e Biomedicina, ramo de Biologia Molecular, Celular e do Desenvolvimento apresentada ao Instituto de Investigação Interdisciplinar da Universidade de Coimbra
Neuronal migration is a fundamental step during brain development. Indeed, the impairment of neuronal migration is one of the major causes of cortical malformation, which has been associated with several neurological and psychiatric disorders. Therefore, it is of upmost importance to unravel the mechanisms driving neuronal migration. In this regard, it was recently shown that adenosine type 2A receptor (A2AR) controls interneuron migration and insertion into the hippocampal circuitry. The work developed during this thesis aimed to evaluate if A2AR is also involved in the migration of cortical principal neurons. For that purpose, it was first evaluated the impact of the genetic deletion (A2AR-KO) or the pharmacological blockade of A2AR on mice cortical neurons migration during embryonic development. In comparison to their wild-type littermates, embryos lacking the A2AR showed impaired migration of cells labelled with BrdU at embryonic day 14.5 (E14.5) and traced at E17.5. Similarly, embryos exposed to the A2AR antagonist SCH58261 (daily 0.1mg/kg intraperitoneal (i.p.) injection in pregnant females from E13.5 to E16.5) displayed impaired migration when compared with embryos exposed to vehicle. This should be due to A2A receptors expressed by migratory neurons since in utero electroporation (IUE) of a plasmid encoding shRNA specific for A2AR (E14.5-E17.5) also impaired migration. The impairment of neuronal migration occurs mostly in the intermediate zone (IZ), where it was observed an accumulation of neurons. Nowadays, it is well-known that there are two fundamental steps for neurons at the IZ to be able to proceed their migration into the cortical plate (CP). It is required a transition from a multipolar to a bipolar shape, followed by the establishment of an axon-like leading process. Accordingly, in cultured mice cortical neurons, it was found that the pharmacological blockade of A2AR with the selective antagonist SCH58261 (50 nM) from day in vitro 0 (DIV0) leads not only to a reduction of the number of axons (SMI-31 positive neurites) but also of their length, analysed at DIV3. This was also appraised in vivo, as it was observed that the knockdown of A2AR led to an impairment of both neuronal polarization and axonal elongation in migratory neurons as well. Furthermore, it was also observed a similar impairment of cell migration in the CD73-KO mice, which lacks the ecto-5'-nucleotidase (e-5NT) that converts extracellular AMP into adenosine, indicating that the adenosine that is activating the A2AR derives from the extracellular catabolism of ATP. This is further heralded by the observation of immunoreactivity for the vesicular nucleotide transporter (VNUT) in mice developing cortex at E13.5 and E17.5. Altogether, these results show that A2A receptors activated by ATP-derived adenosine are involved in the migration of cortical principal neurons, in particular for the transition from the IZ into the CP by controlling the establishment of neuronal polarity and axonal elongation. In order to determine if the ability of A2A receptors to control axon formation is selective or not to cortical neurons, it was performed the same evaluation in cultured rat hippocampal neurons. It was found that the pharmacological activation of A2AR with the selective agonist CGS21680 (30 nM) induced the formation of abnormal secondary axons and neither the blockade or activation of A2A receptors interfered with the formation of the normal axon. These results show that A2AR is also able to control axon formation in hippocampal neurons. In contrast to the observed in cortical cells, the pharmacological blockade of A2A receptors did not inhibit axon formation of rat hippocampal neurons, but instead it is the activation of these receptors that interfered with axon formation, inducing the formation of aberrant secondary axons. Thus, there is a remarkable difference in the control of axon formation by A2AR in cortical and hippocampal neurons. While in cortical neurons there is a tonic regulation of axon formation by A2AR, in hippocampal neurons these receptors are not required for the normal formation of the axon. Furthermore, preliminary results showed that this should be attained by a modulation of collapsin response mediator protein-2 (CRMP-2) phosphorylation, a microtubule-associated protein that plays a crucial role in neurodevelopment, either in axon specification, guidance and outgrowth, and in cortical principal neuron migration. In contrast with the peripheral nervous system (PNS), which demonstrates a remarkable axonal regeneration ability, in the adult mammalian central nervous system (CNS) it is notorious that, after injury, damaged axons are mostly unable to regenerate, leading to the impairment of normal function. Therefore, the comprehension of the differences between those two systems may provide novel potential therapeutic strategies for axon repair. In that sense, during this work it was also explored the role of purinergic receptors in nerve regeneration, in particular the metabotropic P2Y1 receptor (P2Y1R) and A2AR, since it is expected a rapid increase in extracellular ATP levels and its metabolites upon nerve injury. There are systems that have the capacity to self-regenerate after injury, being excellent models to comprehend the mechanisms underlying regeneration. Of these, the sciatic nerve lesion represents a commonly used approach to evaluate naturally occurring mechanisms of regeneration after damage and here, it was used to address the involvement of purinergic receptors during this process. The preliminary results obtained reveal that in the presence of MRS2500 (0.8 mM), a P2Y1R antagonist, it was observed a decrease of 50.6% in the number of myelinated fibres recovered. On the other hand, the antagonism of A2AR did not significantly modify the number of regenerated myelinated fibres. Though, it remains to elucidate if it is the regeneration or the myelination of fibres that are being compromised after P2Y1R blockade. To answer this question, it is necessary to evaluate the number of demyelinated and non-myelinated fibres as well. Altogether, this thesis provides evidences supporting the involvement of A2A receptors in the migration of cortical principal neurons through the modulation of neuronal polarization and axonal elongation, two cellular events fundamental for neuronal migration, most likely through the de-repression of CRMP-2. The impact of this work may be beyond the contribution to the understanding of the physiology of development, since caffeine, the psychoactive drug most consumed in the world, is an antagonist of A2A receptors.
A migração neuronal é um passo fundamental no desenvolvimento cerebral. A diminuição ou bloqueio da migração neuronal são algumas das principais causas de má formação cortical e têm vindo a ser associadas a diversas patologias neurológicas e psiquiátricas. É, portanto, importante desvendar os mecanismos que regem a migração neuronal. Recentemente foi demonstrado que os recetores de adenosina do tipo 2A (A2AR) controlam a migração e inserção dos interneurónios na rede de neurónios do hipocampo. O trabalho que deu corpo a esta tese visou avaliar se os A2AR desempenham também um papel na migração dos neurónios corticais principais. Para este fim, tentou avaliar-se em primeiro lugar o impacto da eliminação genética (A2AR-KO) ou o bloqueio farmacológico dos A2AR nos neurónios corticais principais de murganho durante o desenvolvimento embrionário. Quando comparados com a restante ninhada wild-type, os embriões sem expressão dos A2AR apresentaram alteração da migração celular em células marcadas com BrdU ao dia embrionário 14.5 (E14.5) e analisadas a E17.5. De igual modo, embriões expostos ao antagonista dos A2AR, SCH58261 (injeção intraperitoneal (i.p.) de 0.1mg/kg/dia em fêmeas gestantes desde E13.5 a E16.5), apresentaram alteração da migração neuronal quando comparados com embriões expostos apenas ao veículo. Provavelmente, isto ocorre devido aos A2AR expressos pelos neurónios em migração, já que a eletroporação in utero (IUE) de um plasmídeo que expressa para um shRNA seletivo para os A2AR (E14.5-E17.5) também afetou a migração. Esta alteração na migração neuronal ocorreu principalmente na zona intermédia (IZ), onde se observou uma acumulação neuronal. Atualmente, sabe-se que existem dois passos fundamentais para que os neurónios na IZ prossigam a sua migração para a placa cortical (CP). É necessária uma transição de uma forma multipolar para uma bipolar, seguida do aparecimento de um prolongamento similar ao que dá origem ao axónio. Do mesmo modo, em culturas de neurónios corticais de murganhos foi observado que o bloqueio farmacológico dos A2AR com o antagonista seletivo SCH58261 (50 nM), desde o dia in vitro 0 (DIV0), conduz não só à redução do número de axónios (neurites positivas para SMI-31) como também do seu comprimento, avaliado a DIV3. Observações in vivo levaram a conclusões similares, uma vez que se observou que a eliminação dos A2AR conduziu a uma alteração quer da polarização, quer do alongamento axonal em neurónios em migração. Adicionalmente, foi observada uma alteração na migração celular em murganhos CD73-KO, que não expressam a enzima ecto-5'-nucleotidase (5'-NT) que converte AMP em adenosina, o que indica que a adenosina que ativa os A2AR provêm do catabolismo extracelular do ATP. Esta hipótese é apoiada pela observação de imunoreactividade para o transportador nucleotídico vesicular (VNUT) a E13.5 e a E17.5 no córtex de murganhos em desenvolvimento. Em conjunto, estes resultados demonstram que os recetores A2A ativados pela adenosina derivada do ATP estão relacionados com a migração dos neurónios corticais principais, em particular durante a transição da IZ para a CP, através da regulação da polaridade neuronal e alongamento axonal. Para determinar se a capacidade dos A2AR para controlar a formação do axónio é seletiva dos neurónios corticais, o mesmo tipo de análise foi efetuada em culturas de neurónios de hipocampo de murganho. Foi observado que a ativação farmacológica dos A2AR com o agonista seletivo CGS21680 (30 nM) induziu a formação de axónios secundários aberrantes, mas nem o bloqueio nem a ativação dos A2AR interferiram com a formação do axónio principal. Estes resultados mostram que os A2AR estão também envolvidos no controlo da formação de axónios nos neurónios do hipocampo. Ao contrário do observado nas células corticais, o bloqueio farmacológico dos A2AR não inibiu a formação de axónios nos neurónios do hipocampo de murganho, mas a ativação destes recetores alterou a formação axonal, induzindo o aparecimento anómalo de axónios secundários. Desta forma, existe uma diferença notável no controlo mediado pelos A2AR na formação de axónios nos neurónios corticais e do hipocampo. Enquanto que, em neurónios corticais há uma regulação tónica da formação de axónios pelos A2AR, em neurónios do hipocampo estes recetores não são necessários à formação do axónio principal. Adicionalmente, resultados preliminares mostram que esta formação pode ser conseguida pela modulação da fosforilação da proteína mediadora de resposta da colapsina-2 (CRMP-2), uma proteína associada aos microtúbulos que desempenha um papel crucial no neuro desenvolvimento, tanto na especificação, condução e crescimento do axónio, como na migração de neurónios corticais. Em contraste com o sistema nervoso periférico (SNP), que revela uma marcada capacidade de regeneração axonal, no sistema nervoso central (SNC) do mamífero adulto é notório que, após lesão, os axónios danificados são predominantemente incapazes de regenerar, ficando funcionalmente comprometidos. A compreensão das diferenças entre estes dois sistemas pode, portanto, permitir identificar estratégias terapêuticas inovadoras para a reparação dos axónios. Desta forma, durante este trabalho foi também explorado o papel dos recetores purinérgicos na regeneração nervosa, em particular o recetor metabotrópico P2Y1 (P2Y1R) e o A2AR, uma vez que se antecipa um aumento súbito dos níveis de ATP extracelular, bem como dos seus metabolitos após lesão nervosa. Os sistemas com capacidade regenerativa apresentam-se como modelos de excelência para compreender os mecanismos subjacentes a essa regeneração. De entre eles, a lesão do nervo ciático é um método comum para avaliar os mecanismos de regeneração despoletados pelo dano, que foi usado neste trabalho para abordar o envolvimento dos recetores purinérgicos nesse processo. Os resultados preliminares obtidos demonstraram que na presença de MRS2500 (0.8 mM), um antagonista dos P2Y1R, ocorreu uma diminuição de 50.6% no número de fibras mielinizadas recuperadas. Por outro lado, o antagonismo dos A2AR não revelou diferenças estatisticamente significativas no número de fibras mielinizadas regeneradas. É necessário ainda determinar se o bloqueio dos P2Y1R está a comprometer a regeneração ou a mielinização das fibras. A resposta a esta questão requer a avaliação do número de fibras desmielinizadas e não-mielinizadas. No seu conjunto, esta tese fornece indícios que suportam o envolvimento dos A2AR na migração dos neurónios corticais principais através da modulação da polarização neuronal e alongamento axonal, dois processos celulares fundamentais para a migração neuronal, muito provavelmente pela de-repressão da CRMP-2. O impacto deste trabalho pode ainda estender-se além da compreensão da fisiologia do desenvolvimento, dado que a cafeína, a substância psicoativa mais consumida no mundo, é um antagonista dos A2AR.

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia
Adenosine triphosphate (ATP) is best known as the key molecule in bioenergetics and in multiple cellular processes, but it is now well-recognized as an extracellular signalling molecule as well. ATP can signal directly through the activation of P2Rs or indirectly through P1Rs activated by adenosine formed upon the extracellular catabolism of ATP by ecto-nucleotidases. In the adult brain, purinergic receptors display a widespread regional and cellular distribution, fulfilling a neurotransmitter and neuromodulatory role, controlling astrocytic function or formatting microgliaresponsiveness. Purinergic signaling also plays a role in brain development ranging from neurogenesis to neuronal migration, neuritogenesis and synaptogenesis. Particularly regarding neuronal development, it has been shown that P2Rs bidirectionally regulates axonal growth, promoting axonal elongation through P2Y1R and inhibiting it through P2Y13R and P2X7R. A2ARs also promotes axonal growth and dendritic branching in ratcortical neurons. Unpublished data from our group showed that A2ARs is not only involved in axonal growth but also in axon formation. This was observed both in vitro inmice cortical neurons and in vivo, since it is required for cortical principal neurons migration in the transition from the intermediate zone (IZ) to the cortical plate (CP).The bidirectional regulation of axonal growth by P2Rs was shown to be mediated by a convergent but differential regulation of adenylate cyclase 5 and PI3K-Akt-GSK3βpathway. The A2AR-driven axonal outgrowth in rat cortical neurons was also shown to involve PI3K. Yet, how the purinergic receptors are able to control both dendritic and axonal development remains ill-defined. One protein that might be targeted and mediating the effects of purinergic receptors in neuronal development is collapsin response mediator protein 2 (CRMP2), a microtubule-associated protein shown to contribute to dendritogenesis and axon specification and outgrowth, and critical in cortical principal neurons migration, precisely in the IZ-CP transition. CRMP2 is negatively regulated by phosphorylation by two independent pathways, one mediated by cyclin-dependent kinase (Cdk5) that phosphorylates S522, priming the phosphorylation of T514 by GSK3β, and the other mediated by Rho kinase at T555, also a target of PKC. Thus, to start addressing the putative involvement of CRMP2 regulation in the effects induced by purinergic receptors, it was necessary to evaluate the involvement of each of these kinases namely PKC, GSK3 and Rho kinase.E18-derived rat hippocampal neurons cultured in the presence of the selective A2AR agonist, CGS21680 (30 nM), from day in vitro 0 (DIV0) to DIV3 displayed an increase in the number of axons per neuron and longer axons, an effect not observed in cellselectroporated with shRNA-A2AR. Thus, in contrast to the observed in mice cortical neurons, where A2AR is tonically necessary for the formation of the normal axon, in rathippocampal neurons A2ARs are not involved in the formation of the normal axon.Instead, the exogenous activation of A2AR induces the formation of secondary axons. Moreover, A2ARs activation decreased dendritic length. Preliminary data indicate that all these effects depend on BDNF activity. Furthermore, we observed that P2Y1R does notcontrol axon formation, but it promotes axon outgrowth. The agonist of P2Y1, P2Y12 and P2Y13 receptors, ADPβS (5 μM), and the P2Y1R selective agonist, MRS2365 (100 nM), induced an increase in axon length, while the P2Y1R antagonist, MRS2179 (10 μM), caused a decrease in axon length, showing a tonic action of P2Y1R controlling axon outgrowth. The pharmacological blockade of P2X7R with BBG (100 nM) increased axonal length, indicating that P2X7R is tonically inhibiting axonal growth also in rat hippocampalneurons whilst not modifying dendritic morphology, as previously reported.All these effects were then evaluated in the presence of the kinase inhibitors. Itwas found that A2AR-driven axon elongation involves GSK3, whereas axon formation induced by A2AR was prevented in the presence of PKC and Rho kinase inhibitors. The inhibition of dendritic growth by A2ARs activation depends on PKC and Rho kinase. P2Y1Rpromotes axonal outgrowth through a PKC- and GSK3-dependent mechanism, whereas the inhibition of dendritic growth involves PKC- and Rho kinase.The identification of the enzymes involved in the control of dendritic and axonal development by A2AR- and P2Y1R - support and set the grounds to characterize the involvement of CRMP2 and hence fully identify the signaling pathways recruited by purinergic signaling in the control of neuronal development. Besides, this study also revealed region-specificity in the control of neuronal development by purinergic signalling that is now mandatory to be fully elucidated.
Adenosina trifosfato (ATP) tem um papel central como molécula energética e em múltiplos processos celulares, mas também desempenha um papel na sinalização extracelular. O ATP pode sinalizar ou pela ativação direta dos recetores P2 (P2Rs) ou através dos recetores P1 (P1Rs) ativados pela adenosina formada a partir do catabolismo extracelular de ATP pelas ecto-nucleotidases. No cérebro adulto, os recetores purinérgicos apresentam uma distribuição generalizada, contribuindo para afisiologia sináptica astrocítica e da microglia. A sinalização purinérgica também tem um papel no desenvolvimento do cérebro, nomeadamente na neurogénese, migração neuronal, neuritogénese e sinaptogénese. Em relação ao desenvolvimento neuronal, foi mostrado que os P2Rs podem regular bidireccionalmente o crescimento axonial, aumentando através dos P2Y1R e inibindo através dos P2Y13R e P2X7R. Os A2AR também promovem o crescimento axonial e a ramificação das dendrites em neurónios corticais de rato. Resultados não publicados do nosso grupo mostram que os A2AR estão também envolvidos na formação do axónio. Isto foi observado quer in vitro em neurónios corticais de ratinho, quer in vivo, sendo um processo necessário para a migração dos neurónios corticais principais na transição da zona intermédia (IZ) para a placa cortical (CP).Relativamente às vias de sinalização envolvidas, foi mostrado que a regulação bidirecional do crescimento axonial pelos P2Rs é mediada pela regulação convergente mas diferencial da adenilato ciclase 5 e da via PI3K-Akt-GSK3β. Foi mostrado também que o crescimento promovido pelos A2AR envolve PI3K. Contudo ainda não se sabe qual o(s) mecanismo(s) a jusante que medeia(m) o controlo por parte dos recetores purinérgicos do desenvolvimento das dendrites e axónios. Uma proteína que pode estar envolvida é a proteína collapsin response mediator protein 2 (CRMP2), uma proteínaassociada aos microtúbulos envolvida na dendritogénese e na especificação e crescimento axonial, e essencial na migração dos neurónios corticais principais, precisamente na transição IZ-CP. A CRMP2 é regulada negativamente por fosforilação através de duas vias independentes, uma mediada pela enzima cdk5 que fosforila o resíduo S522, que permite a fosforilação de T514 pela GSK3β, e a outra mediada pela Rho cinase no resíduo T555, que também pode ser fosforilada pela PKC. Para avaliar o possível envolvimento da CRMP2, foi neste estudo avaliado o envolvimento de cada um destas enzimas cinases, nomeadamente PKC, GSK3 e Rho cinase, nos efeitos induzidos pelos recetores purinérgicos.Em culturas primárias de neurónios de hipocampo de rato derivados de embriões E18, a exposição ao agonista seletivo dos A2AR, CGS21680 (30 nM), desde o dia in vitro 0 (DIV0) até DIV3 induziu um aumento no número de axónios e no seu comprimento, um efeito que não foi observado em neurónios eletroporados com shRNA-A2AR. Portanto, ao contrário do observado em neurónios corticais de ratinho, os A2AR não estão envolvidos na formação do axónio normal em neurónios de hipocampo de rato. Em vez disso, a ativação exógena dos A2AR promove a formação de axónios secundários. Em relação às dendrites, a ativação dos A2AR diminuiu o o seu comprimento. Resultados preliminares mostram que todos estes efeitos dependem de BDNF. Em relação ao P2Y1R, os resultados obtidos indicam que este recetor não controla a formação do axónio, mas promove o seu crescimento. O agonista dos recetores P2Y1,12,13, ADPβS (5 μM), e o agonista seletivo de P2Y1, MRS2365 (100 nM), induziram um aumento no comprimento do axónio, enquanto que o bloqueio farmacológico dos P2Y1R, com MRS2179 (10 μM), induziu uma diminuição no comprimento do axónio, demonstrando uma ação tónica dos P2Y1R no crescimento axonial. O bloqueio farmacológico do P2X7R com BBG (100 nM) aumentou o comprimento dos axónios, indicando uma inibição tónica do crescimento axonial, nãotendo modificado a morfologia das dendrites.Relativamente ao envolvimento das diferentes enzimas, os resultados demonstraram que o crescimento axonial induzido pela ativação dos A2AR envolve GSK3, enquanto que a formação de axónios secundários foi prevenida pela presença de inibidores da PKC e da Rho cinase. A inibição do crescimento das dendrites induzido pelo A2AR depende de PKC e Rho cinase. O P2Y1R promove o crescimento axonial por um mecanismo dependente de PKC e GSK3, enquanto que a inibição do crescimento das dendrites envolve PKC e Rho cinase.A identificação destas enzimas envolvidas no controlo do desenvolvimento de dendrites e axónios pelos A2AR e P2Y1R suportam a hipótese de haver um envolvimento de CRMP2 no controlo do desenvolvimento neuronal pelos recetores purinérgicos. Para além disso definem como poderá ser feita essa regulação, que terá que ser avaliadaexperimentalmente. Além disso, este estudo revelou que existem diferenças no controlo do desenvolvimento neuronal pelo sistema purinérgico em diferentes regiões que também será importante elucidar.
Outro - Esta dissertação foi financiada pelo Fundo Europeu de Desenvolvimento Regional (FEDER), através do Programa Operacional Regional Centro 2020 sob o projeto CENTRO-01-0145-FEDER-000008: BrainHealth2020, através do COMPETE 2020 - Programa Operacional Competitividade e Internacionalização e através de fundos nacionais via FCT - Fundação para a Ciência e a Tecnologia sob projetos POCI-01-0145-FEDER-028160 e UID/NEU/04539/2019.

Mitochondrial adaptation in skeletal muscle is promoted by a diverse array of stimuli, and changes in mitochondrial plasticity have been noted as a result of a many exercise modalities. High-intensity interval training is one such modality that promotes mitochondrial adaptation in response to repeated short-duration bouts of intense effort. Another result of intense muscular effort is a decrease in muscle pH, resulting in intracellular acidosis. The effect of this acidosis on oxygen consumption in muscle has received attention previously, with mixed findings. An aspect of skeletal muscle mitochondrial function that has received limited attention is the production of reactive oxygen species. To date a small number of studies have also provided evidence that attenuating the development of intracellular acidosis may have beneficial effects for mitochondrial adaptation. This thesis aimed to further investigate the effect of acidosis on mitochondrial function, and on intracellular signalling for mitochondrial biogenesis.