Calcium Sensors for Functional Magnetic Resonance Imagin

描述(由申请人提供)：该项目的总体目标是开发一套造影剂并进行体内测试，这将使MRI能够对神经元钙水平进行非侵入性成像。这些试剂将提供快速、直接、潜在的细胞分辨率的动态大脑活动读数。新的MRI钙传感器将对大脑研究产生重大影响，无论是通过应用于动物模型中的神经疾病研究，还是作为基础神经科学中的神经网络功能分析工具。这个实验室的长期目标包括在转基因啮齿动物身上应用新的传感器来解剖与学习和记忆有关的神经回路。根据这项建议开展的工作将为今后的生物学研究建立一个方法学平台。我们将合成和测试的造影剂利用了超顺磁性氧化铁纳米颗粒(SPIO)作为磁共振成像显像剂的独特效力。传感器是由钙传感器蛋白连接到SPIO上形成的；在钙的存在下，颗粒聚集在一起，并在T2加权图像中产生巨大的MRI强度变化。将钙调素(CaM)及其底物多肽M13连接到两组纳米粒子上，形成了一种原型传感器，该传感器的EC50为0.8mU/MCa，并伴有较大的MRI信号变化，但该传感器需要进行一些修饰才能实现细胞内的钙传感。在特定目标1中，我们建议对CaM/M13相互作用界面进行计算建模和定点突变，以降低传感器与细胞蛋白质发生交叉反应的可能性。在具体目标2中，我们建议合成新的超小纳米氧化铁颗粒(直径20 nm)，它将对钙浓度的变化做出快速反应。我们的原型钙传感器的修订版结合了AIMS 1和2的结果，将是进一步体内研究的理想选择。在具体目标3中，我们建议测试新的纳米颗粒传感器在细胞中的钙反应，首先通过注射到非洲爪哇卵母细胞，然后通过注射到苍蝇神经元--我们实验室之前的工作表明，苍蝇是一个很好的神经成像试剂测试系统，因为它易于操作，神经元较大，没有血液动力学影响。这些拟议的磁共振钙传感器在哺乳动物大脑中的非侵入性传递和应用超出了这一提议的范围，但构成了我们发展道路上的又一步。

DESCRIPTION (provided by applicant): The overall goal of this project is the development and in vivo testing of a set of contrast agents that will allow noninvasive imaging of neuronal calcium levels by MRI. These reagents will provide a rapid, direct, and potentially cellular-resolution readout of dynamic brain activity. The new MRI calcium sensors will have a great impact on brain research, both through applications to the study of neurological disease in animal models, and as tools for the analysis of neural network function in basic neuroscience. The long-term objectives of this laboratory include applying the new sensors in transgenic rodents to dissect neural circuitry involved in learning and memory. Work performed under this proposal will establish a methodological platform for future biological studies. The contrast agents we will synthesize and test take advantage of the unique potency of superparamagnetic iron oxide nanoparticles (SPIOs) as imaging agents in MRI. Sensors are formed by conjugating calcium sensor proteins to the SPIOs; in the presence of calcium, the particles aggregate and produce large MRI intensity changes in T2-weighted images. A prototype sensor has been formed by conjugating calmodulin (CaM) and its substrate peptide M13 to two populations of nanoparticles; calcium-dependent aggregation with an EC50 of 0.8 mu/M Ca was observed along with large MRI signal changes, but some modifications of this sensor are required for calcium sensing in cells. In Specific Aim 1, we propose computational modeling and site-directed mutagenesis of the CaM/M13 interaction interface to reduce the sensor's potential for cross-reactivity with cellular proteins. In Specific Aim 2, we propose synthesis of new ultrasmall iron oxide nanoparticle conjugates (diameter<< 20 nm) which will respond quickly to changes in calcium concentration. A revision of our prototype calcium sensor that incorporates results of Aims 1 and 2 will be ideal for further studies in vivo. In Specific Aim 3, we propose to test the calcium responses of new nanoparticle sensors in cells, first by injection into Xenopus oocytes, and then by injection into blowfly neurons-previous work from our laboratory showed that the blowfly is a good test system for neuroimaging agents because of its ease of handling, large neurons, and absence of hemodynamic effects. Noninvasive delivery and applications of these proposed MRI calcium sensors in mammalian brains are beyond the scope of this proposal, but constitute a further step in our trajectory.