Extracellular adenosin-5'-triphosphate (ATP) is an important signalling molecule. Cells of eukaryotic tissues release ATP and express responding purinergic receptors. Ionotropic P2X receptors are trimeric ion channels permeable for K+, Na+ and Ca2+ ions. Each subunit consists of two transmembrane domains (TM1 and TM2), an extracellular loop and intracellular N- and C- termini. The transmembrane region is formed by six helical domains. According to the known crystal structure of zfP2X4 receptor, TM1 helixes are oriented peripherally and stabilize TM2 helixes which form the ion gate. However, eletrophysiological studies revealed that TM1 might also participate in channel gating and forming of the ion pore in the open state. The aim of this work was to investigate the role of TM1 in the process of desensitization of rat P2X4 receptor using cystein-scanning mutagenesis. Mutation of two residues (in Asn32 and Tyr42) prolonged desensitization of P2X4 receptor. Moreover, experiments with a partial agonist α,β-methylenadenosin-5'-triphosphate (αβ-meATP) proved that conformation change of TM domains in the process of desensitization is independent on conformation change caused by an agonist binding. Conserved residue Tyr42 is located in the proximity of TM2 of neighbouring subunit. It probably interacts with Met336...

Disordered communication between cortical neurons and glia underlies many of the characteristics of Fragile X syndrome (FXS), the most common monogenic form of intellectual disability and autism spectrum disorder (ASD). Despite extensive research, no effective treatments exist to comprehensively mitigate ASD- or FXS-related cognitive and motor disabilities, sensory hyperresponsivity, seizures, and other excitation-related symptoms. Glial-glial and glial-neuronal communication can be facilitated by purinergic signalling pathways, which utilize ATP, UTP, and their metabolites to influence both short-term and longer-term activation. The overall objective of this thesis work was to establish whether purinergic signalling is dysregulated within cortical astrocytes derived from the Fmr1 KO mouse model of FXS, and furthermore, to determine whether astrocyte purinergic dysregulations contribute to aberrant Fmr1 KO neuronal-glial interactions. Collectively, these studies provide the first reported evidence that P2Y receptor-driven purinergic signalling is elevated in Fmr1 KO cortical astrocytes, and suggest that this impacts the formation and activity of neuronal circuitry in a manner consistent with FXS symptomatology. Fmr1 KO cortical astrocyte dysregulations included elevated expression of P2Y2 and P2Y6 purinergic receptors, increased intracellular calcium release following P2Y activation, aberrant levels of intracellular purinergic signalling molecules, and increased ectonucleotidase glycosylation. UTP treatment promoted excess Fmr1 KO astrocyte expression and secretion of the synaptogenic protein TSP-1 to potentially influence neuronal connectivity, as well as increased phosphorylation of transcription factor STAT3 to likely drive cortical immune responses. Both exogenous UTP and the presence of Fmr1 KO astrocyte secretions promoted neurite outgrowth, while Fmr1 KO astrocyte-neuron co-cultures demonstrated elevated neuronal burst frequency that was normalized through chronic and selective P2Y2 antagonism. Together, these findings indicate novel and significant astrocyte P2Y-mediated purinergic upregulations within the Fmr1 KO mouse cortex, and suggest that astrocyte purinergic signalling should be further investigated in the search for innovative FXS treatments. / Thesis / Doctor of Philosophy (PhD) / Autism spectrum disorders (ASDs) have become a serious health concern in recent years due to rapidly rising rates of diagnosis. Despite extensive research, there are still no effective treatments for these disorders of brain development. It is therefore important that we study the cellular events contributing to ASDs in order to design new therapeutic strategies. The most common inherited form of ASD is Fragile X syndrome (FXS), which is characterized by cognitive and motor disabilities, sensory hyperresponsivity, attention deficits, hyperactivity, and seizures. Using the Fmr1 knockout (KO) mouse model of FXS, recent research has shown that many of these symptoms are related to disordered communication between brain cells within the cerebral cortex; specifically, between neurons and the helper-like cells known as astrocytes. One form of cellular signalling that supports this communication is known as the purinergic signalling pathway. Collectively, this thesis work is the first to show that purinergic signalling is increased in Fmr1 KO mouse cortical astrocytes and that it impacts FXS neuronal connections. Specifically, Fmr1 KO cortical astrocytes demonstrated increased communication using purinergic signalling, due to greater expression of P2Y2 and P2Y6 purinergic receptors and altered levels of the molecules that stimulate these receptors. Activation of Fmr1 KO astrocyte P2Y receptors promoted expression of the neuronal connection-forming protein TSP-1 and stimulated additional astrocyte signalling pathways. As a result of these changes, when Fmr1 KO neurons were grown in the presence of Fmr1 KO astrocytes, they grew longer extensions and demonstrated greater activity than wildtype controls, in a manner consistent with the excitation-related symptoms of FXS. Selectively targeting P2Y2-driven purinergic pathways with drug treatments corrected this activity, thereby revealing a potential new therapeutic approach for FXS. Understanding excess astrocyte P2Y-driven purinergic communication within the brain may therefore provide a foundation for the future development of new FXS treatments.

No description available.

ATP is stored in millimolar concentrations within the intracellular medium but may be released to extracellular sites either through the damaged plasma membrane or by means of various transporters. Extracellular ATP or its enzymatic breakdown products, ADP, AMP, and adenosine, may then stimulate a range of membrane receptors (Rs). These receptors are classified as belonging to two types termed P2 or P1. P2Rs can be, in addition, subdivided into the ligand-activated P2X and the G protein-coupled P2Y types. Adenosine acts on the P1 type of receptor. A further classification identifies seven mammalian subtypes of P2X1-7 and eight mammalian subtypes of P2YRs (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2Y14). P1Rs are either positively (A2A, A2B) or negatively (A1, A3) coupled to adenylate cyclase via the respective G proteins. Already, such a high number of receptors suggests that purine-mediated effects at the cellular but especially whole organism level have an immense variability. Whereas P2XRs respond only the ATP, P2YRs are sensitive to ATP/ADP, UTP/UDP, or UDP–glucose. Inspection of some articles in this Special Issue will teach us that the nucleoside guanosine probably possesses a receptor of its own, that nucleotides can be gradually degraded metabolically to functionally active nucleotides/nucleosides (see above), and indirect effects by stimulating the synthesis or decomposition of purines/pyrimidines may also increase functional diversity. Eventually, P2/P1Rs may interact both with each other as well as with other neurotransmitter receptors. It is, of course, important to note that, in many cases, receptor (sub)type-preferential agonists and highly selective antagonists are available for pharmacological analysis.

Satellite glial cells (SGCs) surround the cell bodies of neurons of the peripheral nervous system, including those of the sensory ganglia. Their close apposition to the neuronal soma allows for bi-directional communication between neurons and SGCs, which are thought to regulate neuronal activity. After nerve injury, SGCs in the dorsal root ganglia contribute to neuropathic pain. Although the mechanisms are not fully understood, SGCs show increased coupling via gap junctions, and communicate with the neuron via bi-directional purinergic signaling after nerve injury. The increased coupling between SGCs and neurons may have implications for chronic pain following peripheral nerve injury. In vivo studies suggest that injury through the administration of capsaicin to the sensory nerve endings causes SGCs to be activated and proliferate. We have shown that capsaicin treatment in an in vitro co-culture of sensory neurons and SGCs increased the expression of the proliferation marker, Ki-67 in the glia. Here, we examine whether purinergic signaling plays a role in the promotion of SGC proliferation.

No description available.

Quimioreceptores centrais (QC) e periféricos (QP) são células especializadas em detectar alterações de CO2, O2 e H+, e promover ajustes na ventilação e pressão arterial via sistema nervoso central. Avaliamos aqui a ação da sinalização purinérgica em áreas que apresentam essa propriedade (RTN, C1, NTScom e RPa) durante as respostas cardiorrespiratórias promovidas pela ativação dos quimiorreceptores, e a possível participação de astrócitos. Encontramos evidências que receptores P2 modulam a resposta de QC no RTN, enquanto que receptores P2Y1 e receptores glutamatérgicos, modulam a resposta de QP no C1, e que a sinalização purinérgica na região do NTScom ou na região RPa não contribui para resposta de QC. A manipulação farmacológica de astrócitos do RTN com fluorocitrato, mas não da região do NTScom e RPa, produz alterações respiratórias via receptores P2. Nossos achados evidenciam a importância e contribuem para descriminação dos mecanismos de ação da sinalização purinérgica na região bulbo ventrolateral rostral durante a ativação QC e QP. / Central (CC) and peripherals (PC) chemoreceptors are specialized cells to detect changes in CO2, O2 and H+, and produce adjustments in ventilation and blood pressure via the central nervous system. Here we evaluate the action of purinergic signaling in areas with this property (RTN, C1, commNTS, RPA) during the cardiorespiratory responses elicited by activation of chemoreceptors, and a possible role of astrocytes. We found evidence that P2 receptors modulate CC responses in RTN, while P2Y1 and glutamate receptors modulate PC responses in C1, and that the purinergic signaling in the RPa and commNTS region does not contribute to CC responses. The pharmacological manipulation of the RTN astrocytes, but not commNTS or RPa, with fluorocitrate produces respiratory changes via P2 receptors. Our findings show the importance and contribute to discrimination of the mechanisms of purinergic signaling in the rostral ventrolateral medulla during CC and PC activation.

Quimioreceptores centrais (QC) e periféricos (QP) são células especializadas em detectar alterações de CO2, O2 e H+, e promover ajustes na ventilação e pressão arterial via sistema nervoso central. Avaliamos aqui a ação da sinalização purinérgica em áreas que apresentam essa propriedade (RTN, C1, NTScom e RPa) durante as respostas cardiorrespiratórias promovidas pela ativação dos quimiorreceptores, e a possível participação de astrócitos. Encontramos evidências que receptores P2 modulam a resposta de QC no RTN, enquanto que receptores P2Y1 e receptores glutamatérgicos, modulam a resposta de QP no C1, e que a sinalização purinérgica na região do NTScom ou na região RPa não contribui para resposta de QC. A manipulação farmacológica de astrócitos do RTN com fluorocitrato, mas não da região do NTScom e RPa, produz alterações respiratórias via receptores P2. Nossos achados evidenciam a importância e contribuem para descriminação dos mecanismos de ação da sinalização purinérgica na região bulbo ventrolateral rostral durante a ativação QC e QP. / Central (CC) and peripherals (PC) chemoreceptors are specialized cells to detect changes in CO2, O2 and H+, and produce adjustments in ventilation and blood pressure via the central nervous system. Here we evaluate the action of purinergic signaling in areas with this property (RTN, C1, commNTS, RPA) during the cardiorespiratory responses elicited by activation of chemoreceptors, and a possible role of astrocytes. We found evidence that P2 receptors modulate CC responses in RTN, while P2Y1 and glutamate receptors modulate PC responses in C1, and that the purinergic signaling in the RPa and commNTS region does not contribute to CC responses. The pharmacological manipulation of the RTN astrocytes, but not commNTS or RPa, with fluorocitrate produces respiratory changes via P2 receptors. Our findings show the importance and contribute to discrimination of the mechanisms of purinergic signaling in the rostral ventrolateral medulla during CC and PC activation.

Prostate cancer (PCa) cells predominantly metastasize to bone and the complex crosstalk between PCa cells and osteoblasts (bone-forming cells) and osteoclasts (bone-destroying cells) leads to increased tumour growth and worsening of bone disease. Understanding the mechanisms of PCa bone metastasis can identify the aggressive fraction of PCa resulting in earlier intervention. The ability of PCa cells to express bone cell-specific features, termed osteomimicry, could potentially explain the osteotropic nature of PCa cells. The aim of this study was to determine the role of osteomimicry in the regulation of epithelial-mesenchymal transition (EMT) in bone metastatic prostate cancer cells. It was demonstrated that the osteoblast-specific marker alkaline phosphatase (bone/liver/kidney) (ALPL) was overexpressed in bone metastatic (ARCaPM), compared to non-metastatic (ARCaPE), human PCa cells. Knockdown of ALPL resulted in decreased cell viability, increased cell death and a change from mesenchymal to epithelial morphology in ARCaPM and PC3 cells, and increased CDH1 expression along with decreased migration in ARCaPM cells. Treatment with extracellular ATP also resulted in decreased viability, increased expression of epithelial markers (CDH1, KRT14) and decreased expression of mesenchymal markers (VIM, ZEB1), and reduced expression of ALPL in ARCaPM cells. Small RNA-sequencing identified microRNAs differentially expressed between ARCaPE and ARCaPM PCa cell lines: miR-373 expression was lower in ARCaPM compared to ARCaPE cells and its overexpression in ARCaPM cells resulted in a change to epithelial morphology, increased expression of the epithelial marker CDH1 and decreased expression of the mesenchymal markers VIM and ZEB1. Finally, the development of a high-throughput screening method to identify novel microRNA regulators of osteomimicry was described, which identified two microRNAs miR-199a-5p and miR-212 as positive regulators of ALP activity. Taken together, this thesis describes the identification of ALPL and ATP as novel regulators of epithelial-mesenchymal transition in PCa cells and high-throughput ALP-activity screening as a powerful tool to identify novel microRNA regulators of ALP expression.

Extracellular adenosine-5'-triphosphate (ATP), released from damaged cells or coreleased as a cotransmitter from synaptic vesicles, acts on its plasma membrane receptors termed purinergic. Purinergic P2X receptors are ATP-gated cation channels. To date seven P2X isoforms designated P2X1-7 have been cloned that are organized as trimeric homomers or heteromers. All P2X subunits share a similar structure consisting of a large extracellular loop, two transmembrane domains and intracellular N- and C- termini. An additional structural feature is conserved aminoacids, these include ten conserved cysteine residues in the extracellular loop. All ectodomain cysteines form disulfide bonds which are organized in two areas: three disulfide bridges are localized in the N-termini half and two in the C-termini half at P2X receptor. ATP binding pocket is apparently localized between two neighbouring subunits. The aim of this Diploma Thesis was to examine the relevance of ectodomain cysteine residue and/or disulfide bonds for the expression, function and ATP binding properties of the P2X receptor. All ten, one by one, ectodomain cysteines were substituted by alanines and ATP-induced currents was recorded in HEK293 cells expressing wild-type P2X4 receptor and its mutants. Low responsible or nonfunctional mutants...