Monitoring Neuronal Activity and Inducible Gene Expression using MRI

在过去的二十年里，报告基因对生物医学研究做出了巨大贡献，并彻底改变了许多实验的设计和实施方式。主要应用包括：（i）监测基因表达水平;（ii）研究蛋白质之间的动态分子相互作用;（iii）研究细胞相互作用（例如神经元网络）;（iv）跟踪正常和异常发育、免疫过程和（干）细胞移植治疗中的细胞命运;以及（v）监测基因治疗。可以使用磁共振成像（MRI）非侵入性地检测的报告基因的可用性将是特别有吸引力的，因为它将允许基因表达位点与（高分辨率）解剖结构以及功能信息的共配准。在这个项目中，我们建议测试的假设，神经元激活诱导的基因表达可以使用化学交换饱和转移（CEST）MRI监测。该MRI对比度基于射频饱和从报告子的可交换质子到水的转移。由于可以设计不同的CEST MRI报告基因，使每个报告基因都能对独特的射频做出反应，因此我们的新成像方法应该可以同时对多个基因的表达进行成像。基于CEST的MRI报告基因如富赖氨酸蛋白（LRP）将在诱导型启动子c-fos和组成型巨细胞病毒（CMV）启动子的调控下克隆和表达。虽然CMV启动子驱动恒定水平的基因表达，但c-fos启动子下的基因将仅响应于细胞外刺激而表达。在大脑中，c-fos在神经元中被诱导，以响应各种刺激（如应激和躯体感觉刺激）的激活。将在细胞培养物中体外评价报告基因表达，并在使用慢病毒载体转导至大鼠脑区域后体内评价报告基因表达。在使用红藻氨酸注射和前爪刺激的神经元活化后，CEST MRI测量将与用功能性MRI（fMRI）测量的神经元活性的变化相关。如果这些研究成功完成，将代表一种独特的方式来研究大脑可塑性在体内。在多个基因的差异表达期间的非侵入性CEST MRI可能潜在地导致可视化和识别常见神经系统疾病的基础途径，这可能进一步有助于设计改进的治疗方法。公共卫生相关性：在健康的大脑以及许多大脑疾病中，神经元活动与基因表达的动态变化有关。使用非侵入性技术跟踪多个基因的表达，可以极大地促进对许多神经系统疾病的潜在途径的识别，也可以改善治疗方法。本研究的目的是开发一个非侵入性成像的基因表达与神经元功能在真实的时间一致的平台。将使用专门为磁共振成像（MRI）设计的人工报告基因监测基因表达，并使用功能性MRI（fMRI）监测神经元活动。

DESCRIPTION (provided by applicant): Over the last two decades, reporter genes have contributed tremendously to biomedical research, and have revolutionized the way many experiments are designed and carried out. The major applications include: (i) monitoring the level of gene expression; (ii) investigating dynamic molecular interactions between proteins; (iii) studying cellular interactions (such as neuronal networks); (iv) tracking cell fates in normal and abnormal development, immunological processes, and in (stem) cell transplantation therapy; and (v) monitoring gene therapy. Availability of reporter genes that can be detected non-invasively using magnetic resonance imaging (MRI) would be particularly attractive, since it would allow co-registration of the site of gene expression with (high resolution) anatomical structures as well as with functional information. In this project, we propose to test the hypothesis that neuronal activation-induced gene expression can be monitored using chemical exchange saturation transfer (CEST) MRI. This MRI contrast is based on the transfer of radiofrequency saturation from the reporter's exchangeable protons to water. Since different CEST MRI reporters can be designed such that each will be responsive to unique radiofrequencies, our new imaging approach should allow simultaneous imaging of the expression of multiple genes. CEST-based MRI reporter genes such as lysine rich protein (LRP) will be cloned and expressed under the regulation of the inducible promoter c-fos and the constitutive cytomegalovirus (CMV) promoter. While the CMV promoter drives constant levels of gene expression, the genes under the c-fos promoter will be expressed only in response to an extracellular stimulation. In the brain, c-fos is induced in neurons in response to activation by various stimuli such as stress and somatosensory stimulation. The reporter expression will be evaluated in vitro in cell cultures, and in vivo after transduction into regions of the rat brain using a lentiviral vector. Following neuronal activation using kainic acid injection and forepaw stimulation, the CEST MRI measurements will be correlated with changes in neuronal activity as measured with functional MRI (fMRI). If these studies are completed successfully, it will represent a unique way to study brain plasticity in vivo. Non-invasive CEST MRI during differential expression of multiple genes may potentially lead to visualizing and identifying pathways that underlie common neurological disorders which may further help in designing improved treatments. PUBLIC HEALTH RELEVANCE: In the healthy brain as well as in many brain disorders neuronal activity is associated with dynamic changes in gene expression. Following the expression of multiple genes using noninvasive technologies could tremendously promote the identification of pathways that underlie many neurological disorders and could also lead to improved treatments. The objective of this study is to develop a platform for the noninvasive imaging of gene expression in concert with neuronal function in real time. Gene expression will be monitored using artificial reporters genes designed specifically for Magnetic Resonance Imaging (MRI) and neuronal activity using functional MRI (fMRI).