Browsing by Subject "Purinergic signaling"

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    Detection of Osmotic Shock-Induced Extracellular Nucleotide Release with a Genetically Encoded Fluorescent Sensor of ADP and ATP
    (MDPI, 2019-07-24) Trull, Keelan J.; Miller, Piper; Tat, Kiet; Varney, S. Ashley; Conley, Jason M.; Tantama, Mathew; Pediatrics, School of Medicine
    Purinergic signals, such as extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP), mediate intercellular communication and stress responses throughout mammalian tissues, but the dynamics of their release and clearance are still not well understood. Although physiochemical methods provide important insight into physiology, genetically encoded optical sensors have proven particularly powerful in the quantification of signaling in live specimens. Indeed, genetically encoded luminescent and fluorescent sensors provide new insights into ATP-mediated purinergic signaling. However, new tools to detect extracellular ADP are still required. To this end, in this study, we use protein engineering to generate a new genetically encoded sensor that employs a high-affinity bacterial ADP-binding protein and reports a change in occupancy with a change in the Förster-type resonance energy transfer (FRET) between cyan and yellow fluorescent proteins. We characterize the sensor in both protein solution studies, as well as live-cell microscopy. This new sensor responds to nanomolar and micromolar concentrations of ADP and ATP in solution, respectively, and in principle it is the first fully-genetically encoded sensor with sufficiently high affinity for ADP to detect low levels of extracellular ADP. Furthermore, we demonstrate that tethering the sensor to the cell surface enables the detection of physiologically relevant nucleotide release induced by hypoosmotic shock as a model of tissue edema. Thus, we provide a new tool to study purinergic signaling that can be used across genetically tractable model systems.
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    P2X7 Receptors in Neurodegeneration: Potential Therapeutic Applications From Basic to Clinical Approaches
    (Frontiers Media, 2021-04-06) Territo, Paul R.; Zarrinmayeh, Hamideh; Medicine, School of Medicine
    Purinergic receptors play important roles in central nervous system (CNS), where the bulk of these receptors are implicated in neuroinflammatory responses and regulation of cellular function of neurons, microglial and astrocytes. Within the P2X receptor family, P2X7 receptor is generally known for its inactivity in normal conditions and activation by moderately high concentrations (>100 μM) of extracellular adenosine 5'-triphosphate (ATP) released from injured cells as a result of brain injury or pathological conditions. Activation of P2X7R contributes to the activation and proliferation of microglia and directly contribute to neurodegeneration by provoking microglia-mediated neuronal death, glutamate-mediated excitotoxicity, and NLRP3 inflammasome activation that results in initiation, maturity and release of the pro-inflammatory cytokines and generation of reactive oxygen and nitrogen species. These components of the inflammatory response play important roles in many neural pathologies and neurodegeneration disorders. In CNS, expression of P2X7R on microglia, astrocytes, and oligodendrocytes are upregulated under neuroinflammatory conditions. Several in vivo studies have demonstrated beneficial effects of the P2X7 receptor antagonists in animal model systems of neurodegenerative diseases. A number of specific and selective P2X7 receptor antagonists have been developed, but only few of them have shown efficient brain permeability. Finding potent and selective P2X7 receptor inhibitors which are also CNS penetrable and display acceptable pharmacokinetics (PK) has presented challenges for both academic researchers and pharmaceutical companies. In this review, we discuss the role of P2X7 receptor function in neurodegenerative diseases, the pharmacological inhibition of the receptor, and PET radiopharmaceuticals which permit non-invasive monitoring of the P2X7 receptor contribution to neuroinflammation associated with neurodegeneration.