Extracellular ATP mediates purinergic signaling throughout the nervous system, and purinergic signaling mechanisms have been associated with multiple brain pathologies including stroke, aging-related neurodegenerative diseases, and epilepsy. Therefore, methods to accurately and precisely detect extracellular ATP are essential to the study of its physiological role. To this end, our broad goal is to develop a set of quantitative optical tools to image and study the signaling dynamics of extracellular ATP in real-time at central synapses. In particular, evidence in recent decades suggests that astrocytes modulate synaptic transmission and plasticity through activity-dependent release of gliotransmitters that include extracellular ATP. Spillover of neurotransmitters during synaptic activity can activate G-protein coupled receptors on perisynaptic astrocytes. It is proposed that this activation of astrocyte receptors elicits a calcium response that can lead to release of ATP as a gliotransmitter. Subsequently, the released ATP or its metabolite adenosine can bind to and activate neuronal purinergic receptors, causing either homosynaptic or heterosynaptic neuromodulation. Thus, extracellular ATP released from astrocytes might act as a feedback signal to modulate synaptic efficacy and network behavior. However, there is an unmet need for new analytical tools to measure extracellular ATP, and the limitations of current detection methods have impeded the resolution of important questions regarding the mechanisms and physiological relevance of ATP as a gliotransmitter. To meet this need, the central hypothesis of this proposal is that genetically-encoded fluorescent biosensors can be engineered to sense extracellular ATP with sensor properties suitable for studying physiologically relevant purinergic signaling. We will test our hypothesis in two working aims:
Aim 1 is to engineer cell surface-tethered biosensors to quantitatively image ATP release and clearance;
Aim 2 is to use these new biosensors to measure activity- dependent ATP release and clearance in primary cultures of astrocytes and neurons as well as in brain slices. Upon completion of these aims, we will be able to provide both new optical tools for measuring extracellular ATP and imaging protocols that are of broad use to the purinergic signaling community.

Public Health Relevance

Purinergic signaling occurs across organ systems and is involved in a wide range of processes from immunity to pain to blood flow. Extracellular ATP is one of the primary autocrine and paracrine purinergic signals. Thus, it is essential to develop analytical tools to detect and measure changes in extracellular ATP in order to understand these diverse aspects of physiology.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21NS092010-02
Application #
8995713
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Morris, Jill A
Project Start
2015-02-01
Project End
2017-01-31
Budget Start
2016-02-01
Budget End
2017-01-31
Support Year
2
Fiscal Year
2016
Total Cost
$229,642
Indirect Cost
$79,642
Name
Purdue University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
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Conley, Jason M; Radhakrishnan, Saranya; Valentino, Stephen A et al. (2017) Imaging extracellular ATP with a genetically-encoded, ratiometric fluorescent sensor. PLoS One 12:e0187481
Norcross, Stevie; Trull, Keelan J; Snaider, Jordan et al. (2017) Extending roGFP Emission via Förster-Type Resonance Energy Transfer Relay Enables Simultaneous Dual Compartment Ratiometric Redox Imaging in Live Cells. ACS Sens 2:1721-1729
Rajendran, Megha; Dane, Eric; Conley, Jason et al. (2016) Imaging Adenosine Triphosphate (ATP). Biol Bull 231:73-84