Development of dual-in vivo quantification system for glutamate and glutamine

描述（由申请人提供）：谷氨酸是大脑中的主要兴奋性神经递质。 正确控制多巴胺能神经传递对神经元功能是必不可少的。 兴奋性的异常增加与神经元疾病有关，如癫痫、阿尔茨海默病和帕金森病。 补充谷氨酸到突触前细胞的主要途径之一是谷氨酰胺-谷氨酸穿梭，其由三个步骤组成：由神经胶质细胞摄取谷氨酸，在神经胶质细胞中将谷氨酸转化为谷氨酰胺，以及将谷氨酰胺输出回到神经元细胞。 一些证据表明，谷氨酰胺-谷氨酸穿梭在病理条件下上调，谷氨酸释放增加可能导致癫痫发作。 然而，以前的检测方法一直无法直接证明谷氨酰胺-谷氨酸穿梭的上调和谷氨酸释放的增加。 我们以前开发的谷氨酸传感器，报告谷氨酸浓度的变化，在青色和黄色荧光蛋白之间的荧光共振能量转移（FRET）效率的变化。 这种传感器可以在活细胞中表达，并以光学方式报告谷氨酸的释放。 此外，可以通过将这些传感器直接应用于组织来检测脑切片中的谷氨酸释放，从而提供了一种检测完整组织中谷氨酸瞬变的方法。 作为检测过程的下一步，我们建议开发一系列谷氨酰胺传感器，以确定谷氨酰胺水平对大脑中谷氨酸水平的影响。 结合以前开发的谷氨酸传感器，所提出的系统将是有用的，在检查是否破坏的谷氨酰胺-谷氨酸穿梭神经元疾病的发病有关。 我们有初步的数据表明，从大肠杆菌谷氨酰胺结合蛋白。大肠杆菌中，可以转化为一个高亲和力，高特异性FRET谷氨酰胺传感器。 使用这个原型传感器作为支架，我们将创建谷氨酰胺传感器，适用于在体内测量谷氨酰胺的亚细胞区室是相关的谷氨酰胺-谷氨酸穿梭。 我们还将进一步优化先前开发的谷氨酸传感器用于细胞溶质谷氨酸浓度测量，并评估传感器是否可用作高通量筛选的平台。 此外，我们建议工程师谷氨酰胺和谷氨酸传感器与两个光谱正交FRET巴黎，使我们可以在同一个实验中使用这两个传感器。 该实验设置将使我们能够以非常高的时间分辨率关联谷氨酸和谷氨酰胺浓度变化。 此外，该系统将作为其他密切相关分子（如GABA和谷氨酸）的双重成像的原理证明。 公共卫生相关性：谷氨酸能神经传递的控制对正常的大脑功能如学习和记忆至关重要，但谷氨酸水平如何调节在很大程度上是未知的。 我们建议开发一种新的，同时检测方法来检测谷氨酸和谷氨酰胺，神经递质和谷氨酸的主要前体，在完整的组织。 这一结果将大大有助于了解大脑中谷氨酸的调节，并将对与谷氨酸兴奋性毒性相关的疾病（如癫痫和阿尔茨海默病）的研究产生直接影响。

DESCRIPTION (provided by applicant): Glutamate is the primary excitatory neurotransmitter in the brain. Proper control of glutamatergic neurotransmission is indispensable for neuronal functions. An abnormal increase in excitability is linked to neuronal diseases such as epilepsy, Alzheimer's, and Parkinson's disease. One of the major pathways for replenishing glutamate to the presynaptic cell is the glutamine- glutamate shuttle, which consists of three steps: uptake of glutamate by glia cells, conversion of glutamate into glutamine in the glia cells, and export of glutamine back to neuronal cells. Some evidence suggesting that the glutamine-glutamate shuttle is up-regulated in pathological conditions and that the resulting increase in glutamate release might cause seizures has been reported. However, previous detection methods have been unable to achieve direct proof for the up-regulation of the glutamine-glutamate shuttle and the increase of glutamate release. We previously developed glutamate sensors that report glutamate concentration change as the change in Fluorescent Resonance Energy Transfer (FRET) efficiency between cyan and yellow fluorescent proteins. Such sensors can be expressed in living cells and report the glutamate release optically. Moreover, glutamate release from a brain slice can be detected by directly applying these sensors to the tissue, offering a method to detect glutamate transient in an intact tissue. As the next step in the detection process, we propose to develop a series of glutamine sensors to determine the effect glutamine levels have on the glutamate level in the brain. Combined with previously developed glutamate sensors, the proposed system will be useful in examining whether the disruption of the glutamine-glutamate shuttle is related to the onset of neuronal diseases. We have preliminary data to suggest that a glutamine binding protein from E. coli can be converted into a high-affinity, high-specificity FRET glutamine sensor. Using this prototype sensor as a scaffold, we will create glutamine sensors that are suitable for in vivo measurements of glutamine in the subcellular compartments that are relevant to the glutamine-glutamate shuttle. We will also further optimize the previously developed glutamate sensor for cytosolic glutamate concentration measurement and evaluate if the sensor could be used as a platform for high-throughput screening. Moreover, we propose to engineer glutamine and glutamate sensors with two spectrally orthogonal FRET paris so that we can use both sensors in the same experiment. This experimental setting will allow us to correlate glutamate and glutamine concentration change at very high time resolution. In addition, this system will serve as the proof-of-principle for dual imaging of other closely related molecules such as GABA and glutamate. PUBLIC HEALTH RELEVANCE: The control of glutamatergic neurotransmission is crucial to normal brain functions such as learning and memory, but how the level of glutamate is regulated is largely unknown. We propose to develop a novel, simultaneous detection method to detect glutamate and glutamine, the neurotransmitter and the major precursor of glutamate, in intact tissue. The result will contribute significantly to the understanding of glutamate regulation in the brain and will have a direct impact on the study of diseases that are related to glutamate excitotoxicity, such as epilepsy and Alzheimer's diseases.