Calcium Sensors for Functional Magnetic Resonance Imagin

用于功能磁共振成像的钙传感器

• 批准号: 7140625
• 负责人: Alan Jasanoff
• 金额: $ 22.56万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-30 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7140625
• 关键词: Diptera; Xenopus oocyte; bioengineering /biomedical engineering; biosensor device; brain imaging /visualization /scanning; calcium indicator; functional magnetic resonance imaging; iron oxide; nanotechnology; neuroimaging; neurons; neurophysiology; noninvasive diagnosis; single cell analysis; site directed mutagenesis; small molecule

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.

描述（由申请人提供）：该项目的总体目标是开发和体内测试一套造影剂，通过MRI对神经元钙水平进行无创成像。这些试剂将提供快速、直接和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的神经系统疾病研究，还是作为基础神经科学中神经网络功能分析的工具。本实验室的长期目标包括在转基因啮齿动物中应用新的传感器来解剖涉及学习和记忆的神经回路。根据本提案开展的工作将为未来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（SPIOs）作为MRI成像剂的独特功效。传感器是通过将钙传感器蛋白偶联到spio上形成的；在钙存在的情况下，颗粒聚集并在t2加权图像上产生较大的MRI强度变化。将钙调蛋白（CaM）及其底物肽M13偶联到两个纳米粒子群上，形成了一个原型传感器；观察到钙依赖性聚集，EC50为0.8 mu/M Ca，并伴有较大的MRI信号变化，但该传感器需要进行一些修改才能在细胞中感知钙。在Specific Aim 1中，我们提出了CaM/M13相互作用界面的计算建模和定点诱变，以减少传感器与细胞蛋白质交叉反应的可能性。在Specific Aim 2中，我们提出了一种新的超小氧化铁纳米颗粒缀合物（直径<< 20 nm）的合成方法，它可以快速响应钙浓度的变化。我们的原型钙传感器的修订版包含目标1和目标2的结果，将是进一步体内研究的理想选择。在Specific Aim 3中，我们建议测试细胞中新型纳米颗粒传感器的钙反应，首先通过注射到非洲爪蟾卵母细胞中，然后注射到苍蝇神经元中——我们实验室之前的工作表明，苍蝇是一种很好的神经显像剂测试系统，因为它易于操作，神经元大，没有血流动力学效应。这些MRI钙传感器在哺乳动物大脑中的无创传递和应用超出了本提案的范围，但在我们的发展轨迹中又迈出了一步。