Genetically-encoded optical sensors to study purinergic signaling

 描述(申请人提供)：细胞外ATP介导整个神经系统的嘌呤能信号，嘌呤能信号机制与包括中风、衰老相关的神经退行性疾病和癫痫在内的多种脑部病理有关。因此，准确、准确地检测胞外ATP的方法对于研究其生理作用是至关重要的。为此，我们的广泛目标是开发一套定量光学工具来实时成像和研究中央突触细胞外ATP的信号动力学。特别是，近几十年的证据表明，星形胶质细胞通过活性依赖的释放包括细胞外ATP在内的胶质递质来调节突触传递和可塑性。突触活动中神经递质的溢出可以激活突触周围星形胶质细胞上的G蛋白偶联受体。有人认为，星形胶质细胞受体的这种激活可以引起钙反应，从而导致作为胶质递质的ATP的释放。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，引起同突触或异突触的神经调节。因此，星形胶质细胞释放的细胞外ATP可能作为反馈信号来调节突触效应和网络行为。然而，对新的分析工具的需求还没有得到满足，目前检测方法的局限性阻碍了关于ATP作为神经胶质递质的机制和生理相关性的重要问题的解决。为了满足这一需求，该建议的中心假设是，可以设计基因编码的荧光生物传感器来检测细胞外ATP，其传感器特性适合于研究与生理相关的嘌呤能信号。我们将在两个工作目标中验证我们的假设：目标1是设计细胞表面捆绑的生物传感器来定量成像ATP的释放和清除；目标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原代培养以及脑片中依赖活性的ATP释放和清除。完成这些目标后，我们将能够提供测量细胞外ATP的新光学工具和对嘌呤能信号社区广泛使用的成像方案。

 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.