Calcium Sensors for Functional Magnetic Resonance Imagin

描述（由申请人提供）：本项目的总体目标是开发和体内测试一组造影剂，该造影剂将允许通过MRI对神经元钙水平进行无创成像。 这些试剂将提供一个快速，直接，和潜在的细胞分辨率的动态大脑活动的读数。 新的MRI钙传感器将对大脑研究产生巨大影响，无论是通过应用于动物模型中神经疾病的研究，还是作为基础神经科学中神经网络功能分析的工具。 该实验室的长期目标包括将新的传感器应用于转基因啮齿动物，以解剖涉及学习和记忆的神经回路。 根据该提案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超顺磁性氧化铁纳米颗粒（SPIO）作为MRI成像剂的独特效力。 通过将钙传感器蛋白与SPIO结合形成传感器;在钙存在的情况下，颗粒聚集并在T2加权图像中产生大的MRI强度变化。 通过将钙调素（CaM）及其底物肽M13与两种纳米颗粒结合，形成了一种原型传感器;观察到钙依赖性聚集，其EC 50为0.8 mu/M Ca，沿着较大的MRI信号变化，但需要对该传感器进行一些修改以用于细胞中的钙传感。 在具体目标1中，我们提出了计算建模和定点诱变的CaM/M13相互作用界面，以减少传感器的潜在交叉反应与细胞蛋白质。 在具体目标2中，我们提出合成新的超小氧化铁纳米颗粒缀合物（直径<< 20 nm），其将对钙浓度的变化迅速响应。 我们的原型钙传感器，结合目标1和2的结果的修订将是理想的进一步研究在体内。 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蟾卵母细胞中，然后通过注射到绿头苍蝇神经元中-我们实验室以前的工作表明，绿头苍蝇是一个很好的神经成像剂的测试系统，因为它易于处理，大神经元，并且没有血液动力学效应。 这些建议的MRI钙传感器在哺乳动物大脑中的非侵入性递送和应用超出了本提案的范围，但构成了我们轨迹的进一步发展。

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.