Genetically-encoded optical sensors to study purinergic signaling

用于研究嘌呤能信号传导的基因编码光学传感器

基本信息

批准号：
8995713
负责人：
Mathew Tantama
金额：
$ 22.96万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-02-01 至 2018-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8995713
关键词：
Adenosine Affinity Astrocytes Axon Behavior Binding Biosensor Blood flow Brain Brain Pathology Calcium Cell surface Cells Communication Communities Detection Electrophysiology (science)Engineering Epilepsy Extracellular Space Feedback G-Protein-Coupled Receptors Glutamates Goals Health Image Immunity In Vitro Kinetics Lead Life Locales Measures Mediating Membrane Methods Microglia Nervous system structure Neurodegenerative Disorders Neuroglia Neurons Neurotransmitters Nucleotidases Optics P2X-receptor Pain Photons Physiological Physiology Presynaptic Terminals Process Property Protein Engineering Protocols documentation Purinoceptor Resolution Role Signal Transduction Slice Stroke Synapses Synaptic Transmission Synaptic plasticity System Testing Time Tissues Vertebral column Vesicle Work age related analytical tool animal tissue autocrine body system brain cell cell type extracellular in vivo live cell microscopy meetings multi-photon neuroregulation optical sensor paracrine receptor response sensor tool transmission process

项目摘要

DESCRIPTION (provided by applicant): 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.

描述（由适用提供）：细胞外ATP介导整个神经系统中的嘌呤能信号传导，以及嘌呤能信号传导机制与多种脑病理学有关，包括中风，与衰老相关的神经退行性疾病和癫痫病。因此，准确和精确检测细胞外ATP的方法对于研究其身体作用至关重要。为此，我们的广泛目标是开发一组定量的光学工具，以实时在中央突触时实时对细胞外ATP的信号传导动力学进行图像和研究。尤其是，最近几十年的证据表明，星形胶质细胞通过活动依赖性的胶质递质的释放来调节合成传播和可塑性，包括细胞外ATP。突触活性期间神经递质的Spilover可以激活环突触星形胶质细胞上的G蛋白偶联受体。有人提出，这种星形胶质细胞受体的激活会引起钙反应，该反应可能导致ATP释放为Gliotransmitter。随后，释放的ATP或其代谢产物腺苷可以与神经元嘌呤能受体结合并激活，从而导致同性突触或异质性神经调节。这是从星形胶质细胞释放的细胞外ATP可能是调节突触效率和网络行为的反馈信号。但是，未满足的新分析工具来测量细胞外ATP，并且当前检测方法的局限性阻碍了有关ATP作为Gliotransmitter的机制和物理相关性的重要问题的解决方案。为了满足这一需求，该提案的中心假设是，可以对一般编码的荧光生物传感器进行设计，以感知细胞外ATP，其传感器特性适合研究物理相关的嘌呤能信号传导。我们将在两个工作目的中检验我们的假设：目标1是设计细胞表面螺旋状生物传感器以定量图像ATP释放和清除率；目的2是使用这些新的生物传感器来测量星形胶质细胞和神经元的原发性培养物以及脑切片中的活性依赖性ATP的释放和清除率。这些目标完成后，我们将能够提供新的光学工具来测量对嘌呤能信号群落广泛使用的细胞外ATP和成像协议。