Estudos têm demonstrado que o microambiente tumoral é rico em ATP e adenosina, sugerindo o envolvimento da sinalização purinérgica no desenvolvimento e/ou manutenção do câncer. Ainda, o receptor purinérgico P2X7, conhecido pelo seu papel na indução de apoptose, encontra-se reduzido em alguns tecidos tumorais em comparação aos tecidos saudáveis, indicando que a sua redução possa ser um mecanismo de resistência celular à apoptose. Dessa forma, compreender o papel da sinalização purinérgica no contexto do câncer se torna indispensável e permite que novas abordagens terapêuticas sejam implementadas. Nesse trabalho, avaliamos a função dos nucleotídeos e nucleosídeos da adenina, bem como do receptor P2X7 na indução da morte celular em células de câncer cervical. Também verificamos o efeito do heat shock na potencialização da atividade do receptor P2X7 frente à curta exposição ao ATP em células de câncer de cólon. De acordo com os nossos resultados, o efeito citotóxico do ATP extracelular nas linhagens de câncer cervical é mediado principalmente pela ação do seu metabólito adenosina, que ao entrar no interior das células, promove o aumento dos níveis intracelulares de AMP, ativação de AMPK, aumento da p53 e indução de autofagia. O papel do receptor P2X7 nesse contexto parece ser apenas coadjuvante, visto que o seu bloqueio ou silenciamento impediu em apenas 20% a morte celular. Além disso, utilizando células de câncer de cólon, nós demonstramos que o heat shock aumenta a funcionalidade do receptor P2X7, independente da interação com heat shock proteins ou canais do tipo conexina/panexina, potencializando o efeito citotóxico do ATP. Esse efeito parece estar relacionado à mudanças na composição e arquitetura da membrana celular, visto que o uso do agente fluidizador de membrana benzil álcool foi capaz de mimetizar o efeito do heat shock na potencialização do receptor P2X7 a 37ºC. Este estudo fornece evidências adicionais sobre o papel da sinalização purinérgica no contexto da biologia celular tumoral e abre novas perspectivas para o uso dos nucleotídeos de adenina associados a hipertermia como agentes adjuvantes na terapia do câncer. / The tumor microenvironment is rich in ATP and adenosine, suggesting an involvement for purinergic signaling in cancer development and surveillance. The P2X7 receptor, among the P2 purinergic receptors, is broadly recognized as the “death receptor”, because it promotes cell apoptosis when exposed to high levels of extracellular ATP. Researches have been shown that P2X7 protein levels are decreased at the tumor site in comparison to adjacent healthy tissue, suggesting a mechanism of tumor escape to cell death. Thus, understanding purinergic signaling in a cancer context becomes urgent and opens a new field for therapeutic strategies. Here, we evaluated adenine nucleotides and nucleosides cytotoxicity, as well as P2X7 role in cell death induction using cervical cancer cell lines. Indeed, we investigated heat shock effect on P2X7 functionality through exposing colon cancer cell shortly to ATP at 40ºC. According to our data, adenosine uptake formed from ATP metabolism is the main responsible for the extracellular ATP cytotoxicity in cervical cancer cells. While inside of the cell, adenosine is converted to AMP, leading to AMPK activation, p53 increase and autophagy induction. ATP induced cell death per se through P2X7 in this context seems to be less important, since P2X7 blockage or knocking down reduced only 20% of cell death. In colon cancer cells, we found that heat shock stress was able to increase P2X7 pore formation independently of heat shock protein interaction or native pore-forming transporters association (e.g pannexin-or connexin-type channels), thus leading to an increase ATP cytotoxicity. The mechanism enrolled in this process seems to be related to changes in the lipid composition and architecture of membrane, as the membrane fluidizer benzyl alcohol could reproduce heat stress effect in potentiating P2X7 activation at 37ºC. In conclusion, our work provides further evidence for a purinergic signaling role in the cancer biology context and opens new perspectives for the utility of purine-based drugs associated to hypertermia as adjunctive agents in cancer therapy.

Estudos têm demonstrado que o microambiente tumoral é rico em ATP e adenosina, sugerindo o envolvimento da sinalização purinérgica no desenvolvimento e/ou manutenção do câncer. Ainda, o receptor purinérgico P2X7, conhecido pelo seu papel na indução de apoptose, encontra-se reduzido em alguns tecidos tumorais em comparação aos tecidos saudáveis, indicando que a sua redução possa ser um mecanismo de resistência celular à apoptose. Dessa forma, compreender o papel da sinalização purinérgica no contexto do câncer se torna indispensável e permite que novas abordagens terapêuticas sejam implementadas. Nesse trabalho, avaliamos a função dos nucleotídeos e nucleosídeos da adenina, bem como do receptor P2X7 na indução da morte celular em células de câncer cervical. Também verificamos o efeito do heat shock na potencialização da atividade do receptor P2X7 frente à curta exposição ao ATP em células de câncer de cólon. De acordo com os nossos resultados, o efeito citotóxico do ATP extracelular nas linhagens de câncer cervical é mediado principalmente pela ação do seu metabólito adenosina, que ao entrar no interior das células, promove o aumento dos níveis intracelulares de AMP, ativação de AMPK, aumento da p53 e indução de autofagia. O papel do receptor P2X7 nesse contexto parece ser apenas coadjuvante, visto que o seu bloqueio ou silenciamento impediu em apenas 20% a morte celular. Além disso, utilizando células de câncer de cólon, nós demonstramos que o heat shock aumenta a funcionalidade do receptor P2X7, independente da interação com heat shock proteins ou canais do tipo conexina/panexina, potencializando o efeito citotóxico do ATP. Esse efeito parece estar relacionado à mudanças na composição e arquitetura da membrana celular, visto que o uso do agente fluidizador de membrana benzil álcool foi capaz de mimetizar o efeito do heat shock na potencialização do receptor P2X7 a 37ºC. Este estudo fornece evidências adicionais sobre o papel da sinalização purinérgica no contexto da biologia celular tumoral e abre novas perspectivas para o uso dos nucleotídeos de adenina associados a hipertermia como agentes adjuvantes na terapia do câncer. / The tumor microenvironment is rich in ATP and adenosine, suggesting an involvement for purinergic signaling in cancer development and surveillance. The P2X7 receptor, among the P2 purinergic receptors, is broadly recognized as the “death receptor”, because it promotes cell apoptosis when exposed to high levels of extracellular ATP. Researches have been shown that P2X7 protein levels are decreased at the tumor site in comparison to adjacent healthy tissue, suggesting a mechanism of tumor escape to cell death. Thus, understanding purinergic signaling in a cancer context becomes urgent and opens a new field for therapeutic strategies. Here, we evaluated adenine nucleotides and nucleosides cytotoxicity, as well as P2X7 role in cell death induction using cervical cancer cell lines. Indeed, we investigated heat shock effect on P2X7 functionality through exposing colon cancer cell shortly to ATP at 40ºC. According to our data, adenosine uptake formed from ATP metabolism is the main responsible for the extracellular ATP cytotoxicity in cervical cancer cells. While inside of the cell, adenosine is converted to AMP, leading to AMPK activation, p53 increase and autophagy induction. ATP induced cell death per se through P2X7 in this context seems to be less important, since P2X7 blockage or knocking down reduced only 20% of cell death. In colon cancer cells, we found that heat shock stress was able to increase P2X7 pore formation independently of heat shock protein interaction or native pore-forming transporters association (e.g pannexin-or connexin-type channels), thus leading to an increase ATP cytotoxicity. The mechanism enrolled in this process seems to be related to changes in the lipid composition and architecture of membrane, as the membrane fluidizer benzyl alcohol could reproduce heat stress effect in potentiating P2X7 activation at 37ºC. In conclusion, our work provides further evidence for a purinergic signaling role in the cancer biology context and opens new perspectives for the utility of purine-based drugs associated to hypertermia as adjunctive agents in cancer therapy.

Adulte Neurogenese steht im fokus der Neurowissenschaften. Die Neurogenese im Hippokampus spielt eine wichtige Rolle nach verschiedenen pathophysiologischen Ereignissen, z.B. nach epileptischen Anfällen. ATP ist ein wichtiger extrazellulärer Botenstoff, der auch im Gehirn verschiedene Prozesse beinflusst. Es wurden funktionelle P2X7- und P2Y1-Rezeptore an den Vorläuferzellen nachgewiesen. Unsere Hypothese lautet: nach einen epileptischen Anfall kommt es zur massiven Ausströmung von ATP, welches durch P2Y1-Rezeptore die Neurogenese fördert. Dieser Prozess wird durch die Aktivierung der P2X7-Rezeptoren entegegengesteuert, indem der Zelltod der neuen Zellen eingeleitet wird.

Organotypic hippocampal slice cultures were used to model the effects of neuroinflammatory conditions following an epileptic state on functional P2X7 receptors (Rs) of subgranular zone (SGZ) neural progenitor cells (NPCs). The compound, 4-aminopyridine (4-AP), is known to cause pathological firing of neurons, consequently facilitating the release of various transmitter substances including ATP. Lipopolysaccharide (LPS) and interleukin-1(IL-1) both potentiated the dibenzoyl-ATP (Bz-ATP)-induced current amplitudes in NPCs, although via different mechanisms. Whereas LPS acted via promoting ATP release, IL-1 acted via its own receptor to directly influence P2X7Rs. Thus, the effect of LPS was inhibited by the ecto-ATPase inhibitor, apyrase, but not by the IL-1 antagonist, interleukin-1RA (IL-1RA); by contrast, the effect of IL-1 was inhibited by IL-1RA, but not by apyrase. Eventually, incubation with 4-AP upregulated the number of nestin/glial fibrillary acidic protein/P2X7R immunoreactive cells and their appropriate staining intensity, suggesting increased synthesis of P2X7Rs at NPCs. In conclusion, inflammatory cytokines accumulating after epilepsy-like neuronal firing may facilitate the effect of endogenous ATP at P2X7Rs of NPCs, thereby probably promoting necrosis/apoptosis and subsequent cell death.

Extracellular adenosine 50-triphosphate (ATP) in the brain is suggested to be an etiological factor of major depressive disorder (MDD). It has been assumed that stress-released ATP stimulates P2X7 receptors (Rs) at the microglia, thereby causing neuroinflammation; however, other central nervous system (CNS) cell types such as astrocytes also possess P2X7Rs. In order to elucidate the possible involvement of the MDD-relevant hippocampal astrocytes in the development of a depressive-like state, we used various behavioral tests (tail suspension test [TST], forced swim test [FST], restraint stress, inescapable foot shock, unpredictable chronic mild stress [UCMS]), as well as fluorescence immunohistochemistry, and patch-clamp electrophysiology in wild-type (WT) and genetically manipulated rodents. The TST and FST resulted in learned helplessness manifested as a prolongation of the immobility time, while inescapable foot shock caused lower sucrose consumption as a sign of anhedonia. We confirmed the participation of P2X7Rs in the development of the depressive-like behaviors in all forms of acute (TST, FST, foot shock) and chronic stress (UCMS) in the rodent models used. Further, pharmacological agonists and antagonists acted in a different manner in rats and mice due to their diverse potencies at the respective receptor orthologs. In hippocampal slices of mice and rats, only foot shock increased the current responses to locally applied dibenzoyl-ATP (Bz-ATP) in CA1 astrocytes; in contrast, TST and restraint depressed these responses. Following stressful stimuli, immunohistochemistry demonstrated an increased co-localization of P2X7Rs with a microglial marker, but no change in co-localization with an astroglial marker. Pharmacological damage to the microglia and astroglia has proven the significance of the microglia for mediating all types of depression-like behavioral reactions, while the astroglia participated only in reactions induced by strong stressors, such as foot shock. Because, in addition to acute stressors, their chronic counterparts induce a depressive-like state in rodents via P2X7R activation, we suggest that our data may have relevance for the etiology of MDD in humans.

Epilepsy is one of the most common chronic diseases of the central nervous system (CNS). Treatment of epilepsy remains, however, a clinical challenge with over 30% of patients not responding to current pharmacological interventions. Complicating management of treatment, epilepsy comes with multiple comorbidities, thereby further reducing the quality of life of patients. Increasing evidence suggests purinergic signalling via extracellularly released ATP as shared pathological mechanisms across numerous brain diseases. Once released, ATP activates specific purinergic receptors, including the ionotropic P2X7 receptor (P2X7R). Among brain diseases, the P2X7R has attracted particular attention as a therapeutic target. The P2X7R is an important driver of inflammation, and its activation requires high levels of extracellular ATP to be reached under pathological conditions. Suggesting the therapeutic potential of drugs targeting the P2X7R for epilepsy, P2X7R expression increases following status epilepticus and during epilepsy, and P2X7R antagonism modulates seizure severity and epilepsy development. P2X7R antagonism has, however, also been shown to be effective in treating conditions most commonly associated with epilepsy such as psychiatric disorders and cognitive deficits, which suggests that P2X7R antagonisms may provide benefits beyond seizure control. This review summarizes the evidence suggesting drugs targeting the P2X7R as a novel treatment strategy for epilepsy with a particular focus of its potential impact on epilepsy-associated comorbidities.

P2X7 receptors are members of the ATP-gated cationic channel family with a preferential localization at the microglial cells, the resident macrophages of the brain. However, these receptors are also present at neuroglia (astrocytes, oligodendrocytes) although at a considerably lower density. They mediate necrosis/apoptosis by the release of pro-inflammatory cytokines/chemokines, reactive oxygen species (ROS) as well as the excitotoxic (glio)transmitters glutamate and ATP. Besides mediating cell damage i.e., superimposed upon chronic neurodegenerative processes in Alzheimer’s Disease, Parkinson’s Disease, multiple sclerosis, and amyotrophic lateral sclerosis, they may also participate in neuroglial signaling to neurons under conditions of high ATP concentrations during any other form of neuroinflammation/neurodegeneration. It is a pertinent open question whether P2X7Rs are localized on neurons, or whether only neuroglia/microglia possess this receptor-type causing indirect effects by releasing the above-mentioned signaling molecules. We suggest as based on molecular biology and functional evidence that neurons are devoid of P2X7Rs although the existence of neuronal P2X7Rs cannot be excluded with absolute certainty.

In contrast to mammals, adult zebrafish undergo successful neural regeneration following spinal cord injury (SCI). Radial glia (RG) lining the zebrafish central canal undergo injury-induced proliferation and subsequent neuronal differentiation to replace damaged cells and restore motor function. However, the molecular mechanisms that underlie these processes remain elusive. Here, we demonstrate that signaling through the evolutionarily conserved purinergic P2X7 receptor is involved. Within the zebrafish spinal cord, P2X7 receptors have widespread distribution with specific localization to neurons and radial glia. At the protein level, the predominant P2X7 receptor isoforms in zebrafish did not include the full-length variant expressed throughout the murine central nervous system, but two truncated splice variants. In response to SCI, protein expression of the 50 kDa isoform became downregulated at 7 dpi and returned to basal levels of expression at 14 and 21 dpi when compared to naïve controls. Meanwhile, expression of the 37 kDa isoform did not change following injury. Pharmacological activation of P2X7 following SCI resulted in a greater number of proliferating cells around the central canal by 7 dpi, while P2X7 inhibition appeared to have no effect. At 14 dpi, these treatments did not have a significant effect on the number of neurons within the injured spinal cord. This data indicates that P2X7 receptor activation is sufficient to induce cellular proliferation, but not a necessary mediator of either proliferation or neurogenesis following SCI in adult zebrafish. Our findings suggest that unlike in humans, P2X7 signaling may not play a maladaptive role following SCI in adult zebrafish. / Thesis / Master of Science (MSc) / Spinal cord injury in mammals causes widespread neuronal cell death and paralysis. In comparison, zebrafish regenerate damaged neurons and restore motor function. Radial glial cells within the zebrafish spinal cord maintain stem-cell properties. Following injury, these cells divide and replace motor neurons. Since mammals have similar cell-types within the spinal cord, understanding the molecular cues driving this adaptive response is of great interest. Here, we examined the evolutionarily conserved purinergic signaling system and found that the expression of the P2X7 receptor varies significantly from mammals and promotes radial glia division following injury.

No description available.

Dor abdominal pode ser consequente a inúmeras causas, entre as várias possibilidades precisamos ficar atentos aos quadros de isquemia intestinal. No trato digestório a isquemia/reperfusão intestinal (I/R-i) acarreta alterações morfológicas nos neurônios entéricos. Este trabalho tem como objetivo analisar o comportamento das diferentes classes neuronais e do receptor P2X7 no plexo mioentérico do íleo de ratos Wistar submetidos à I/R-i. A isquemia intestinal foi obtida pela obstrução do fluxo sanguíneo das artérias ileais no período de 35 minutos, seguida pelos períodos de reperfusão de 6, 24, 72 horas e 1 semana. No grupo sham não houve a oclusão das artérias ileais. Os tecidos foram preparados por métodos imunohistoquímicos de duplas marcações do receptor P2X7 com a Óxido Nítrico Sintase neuronal (NOSn), calbindina (Calb), calretinina (Calr) e Acetilcolina Transferase (ChAT) e do receptor P2X7, da NOSn e da ChAT com o pan-neuronal anti-HuC/D. As análises qualitativas e quantitativas das contagens das duplas marcações, das densidades neuronais e da área dos perfis foram obtidas dos microscópios de fluorescência e de Confocal de Varredura à Laser. Os resultados qualitativos demonstraram diminuição da expressão do receptor P2X7 no grupo I/R-i de 24 horas e retorno da expressão nos grupos I/R-i de 72 horas e 1 semana. Os dados quantitativos demonstraram: a) os neurônios do plexo mioentérico foram imunorreativos ao receptor P2X7; b) não houve alterações significativas nas duplas marcações do receptor P2X7 com os neurônios NOSn-ir, Calr-ir, Calb-ir e ChAT-ir nos grupos sham e I/R-i; c) não houve alterações significativas nas duplas marcações do receptor P2X7, e dos neurônios NOSn-ir e ChAT-ir com o pan-neuronal anti-HuC/D nos grupos sham e I/R-i; d) houve diminuição nas densidades nos grupos I/R-i com 6, 24, 72 horas e 1 semana dos neurônios P2X7-ir, NOSn-ir, Calr-ir, Calb-ir, ChAT-ir e anti-HuC/D-ir quando comparados aos grupos sham; e) houve um aumento na área do perfil dos neurônios NOSn-ir nos grupos I/R-i de 6 e 24 horas, nos neurônios ChAT-ir houve um aumento no grupo I/R-i de 1 semana e nos neurônios Calr-ir houve uma diminuição no grupo I/R-i de 6 horas e um aumento no grupo I/R-i de 24 horas quando comparados aos grupos sham. O presente estudo demonstrou que a I/R-i está associada com a perda significativa de diferentes subpopulações de neurônios do plexo mioentérico acompanhada por diversas alterações morfológicas, o que pode acarretar problemas na motilidade intestinal. / Abdominal pain may be consequent to numerous causes, among the various possibilities need to be attentive to intestinal ischemia. In the digestive tract the intestinal ischemia-reperfusion (I/R-i) causes morphological changes in enteric neurons. The aim of the work was to analyze the behavior of different neurons and P2X7 receptor in the myenteric plexus of the ileum of rats subjected to I/R-i. Intestinal ischemia was obtained by the obstruction of blood flow in the ileal arteries period of 35 minutes followed by reperfusion periods of 6, 24, 72 hours and 1 week. In the sham group there was no occlusion of the ileal arteries. The tissues were prepared by immunohistochemical methods for double staining of P2X7 receptor with neuronal nitric oxide synthase (nNOS), calbindin, calretinin and acetylcholine transferase (ChAT) and P2X7 receptor, the nNOS and ChAT with pan-neuronal marker anti-HuC/D The qualitative and quantitative analysis of the counting of double staining, the neuronal density and the area of the cell body profile were obtained from fluorescence microscopy and confocal scanning laser. The qualitative results showed decreased expression of the P2X7 receptor in I/R-i for 24 hours group and return the expression in I/R-i for 72 hours and 1 week groups. The quantitative data showed: a) neurons in the myenteric plexus were immunoreactive for P2X7 receptor; b) no significant changes in the double staining of P2X7 receptor with nNOS, calretinin, calbindin and ChAT neurons in the sham and I/R-i groups; c) does not significant changes in the double staining of the P2X7 receptor, the nNOS and ChAT neurons with the pan-neuronal marker anti-HuC/D in sham and I/R-i groups; d) the densities of the P2X7 receptor, nNOS-IR, calretinin-IR, calbindin-IR, ChAT-IR and anti-HuC/D-IR neurons were decreased in I/R-i 6, 24, 72 hours and 1 week groups, when compared to sham group; e) the profile area was increased in nNOS-IR neurons in the I/R-i for 6 and 24 hours groups, ChAT-IR neurons in I/R-i 1 week group and in the calretinin-IR neurons there was a decrease in I/R-i 6 hours group and an increase in I/R-i for 24 hours group when compared to sham group. The present study demonstrated that I/R-i is associated with significant loss of different subpopulations of neurons in the myenteric plexus accompanied by several morphological changes, which can cause intestinal motility disorder.