Multicolor Fluorescent Sensors for Imaging Zinc Dynamics in Cells

用于细胞内锌动态成像的多色荧光传感器

• 批准号: 8907786
• 负责人: Jacob M. Goldberg
• 金额: $ 5.42万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-23 至 2017-07-22
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8907786
• 关键词: Affect; Affinity; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Apoptotic; Binding; Biochemical; Brain; Brain Part; Calcium Channel; Cell Communication; Cells; Cerebral cortex; Cerebrum; Chelating Agents; Chemicals; Communities; Coumarins; Data; Dendrites; Devices; Discrimination; Disease; Electron Transport; Ensure; Epilepsy; Event; Family; Fluorescein; Fluorescence; Glutamates; Goals; Hippocampus (Brain); Histocompatibility Testing; Homeostasis; Homing; Image; In Vitro; Individual; Information Storage; Ions; Ischemia; Kinetics; Laboratories; Learning; Life; Long-Term Potentiation; Malignant neoplasm of prostate; Mammary gland; Mediating; Memory; Modification; Molecular; Movement; Necrosis; Neurodegenerative Disorders; Neurons; Nitrogen; Odors; Olfactory Pathways; Organelles; Pancreas; Pathology; Pattern; Peptides; Play; Presynaptic Terminals; Process; Property; Prostate; Proteins; Role; Signal Transduction; Smell Perception; Stimulus; Synapses; Synaptic plasticity; System; Testing; Time; Tissue Sample; Tissues; Vesicle; Work; Zinc; base; design; electronic structure; experience; improved; insight; member; neuronal cell body; novel; novel therapeutics; olfactory bulb; postsynaptic; presynaptic; prevent; public health relevance; receptor; research study; resorufin; response; scaffold; sensor; sensory discrimination; small molecule; tool; trafficking; vesicular release; voltage

DESCRIPTION (provided by applicant): Mobile zinc is an important signaling ion in the brain, where it can be found concentrated in certain neurons in the cerebral cortex. Zinc has been shown to modulate long-term potentiation at synapses and is thought to play a role in memory and learning. In healthy individuals, the flow of zinc between neurons and its subsequent intracellular trafficking is tightly regulated but poorly understood. Dysregulation of zinc transpot networks can affect brain excitability. Disruptions in zinc homeostasis have been implicated in a number of pathologies, including Alzheimer's disease, amyotrophic lateral sclerosis (Lou Gehrig's disease), epilepsy, and ischemia. The biochemical basis for zinc's role in these diseases is largely unknown. An improved understanding of how zinc moves into and through neuronal cells will offer valuable insight into how the brain works and might provide clues about the molecular basis underlying these debilitating neurodegenerative diseases. From this information, it may be possible to design new therapeutic strategies. To accomplish these goals, new tools to study zinc dynamics in live cells will be developed. Specifically, a family of multicolor fluorescent sensors with similar zinc binding properties will be synthesized and characterized. The probes will be attached to cellular homing molecules that will direct each probe to a certain compartment or substructure of a cell. The entire set of probes will be applied to a tissue sample and imaged simultaneously. In this manner it will be possible to observe zinc dynamics at multiple organelles over the same timecourse. Application of such probes in neuronal tissue, for example, will allow for the tracking of zinc transport during excitatory event. In this way, new experiments can be designed to rigorously characterize the role of zinc in cell-to-cell communication and in healthy and disease states. The tools developed in the study will be broadly applicable for imaging zinc dynamics in other tissues throughout the body including the mammary gland, pancreas, and prostate, where zinc has been implicated in multiple disease pathologies, such as prostate cancer. In separate but related work, experiments will be performed to test the hypothesis that odor learning depends on synaptic release of zinc in the olfactory bulb. Synaptically released zinc is closely associated with information storage in some parts of the brain by altering plasticity-how the activity of a synapse changes over time in response to a stimulus. Curiously, the only synapses in the olfactory bulb known to show plasticity also have some of the highest concentrations of zinc. Chelators, which are molecules that sequester zinc tightly, and fluorescent zinc indicators will be used to study the role of zincin generating plasticity. Because these synapses appear to be engaged in challenging sensory discriminations, tests will be conducted to probe how zinc release is affected in the olfactory bulb after learning a difficult odor discrimination task. From the data collected in these experiments, one will be able to determine if the patterns of zinc release change upon odor-mediated learning and draw conclusions about the importance of zinc in modulating olfaction.

描述（由申请人提供）：移动锌是大脑中重要的信号离子，可以发现它集中在大脑皮层中的某些神经元中。已显示锌可以调节突触时的长期增强，并被认为在记忆和学习中起作用。在健康的个体中，神经元之间的锌流及其随后的细胞内贩运受到严格调节，但了解不足。锌转盘网络的失调可能会影响大脑兴奋性。锌稳态的干扰与许多病理有关，包括阿尔茨海默氏病，肌萎缩性侧面硬化症（Lou Gehrig的疾病），癫痫和缺血。锌在这些疾病中作用的生化基础在很大程度上是未知的。对锌如何进入神经元细胞的方式有了深入的了解，将为大脑的工作原理提供宝贵的见解，并可能提供有关这些令人衰弱的神经退行性疾病的分子基础的线索。从这些信息可以设计新的治疗策略。为了实现这些目标，将开发用于研究活细胞中锌动态的新工具。具体而言，将合成和表征具有相似锌结合特性的多色荧光传感器家族。这些探针将附着在细胞归巢分子上，该分子将将每个探针引导到细胞的某个隔室或子结构。整个探针将应用于组织样品并同时成像。通过这种方式，可以在相同的时间上观察多个细胞器的锌动力学。例如，这种探针在神经元组织中的应用将允许在兴奋性事件中跟踪锌传输。这样，可以设计新的实验来严格地表征锌在细胞对细胞通信以及在健康和疾病状态中的作用。该研究中开发的工具将广泛适用于整个身体其他组织中的锌动力学成像，包括乳腺，胰腺和前列腺，在该组织中，锌与多种疾病的病理有关，例如前列腺癌。在单独但相关的工作中，将进行实验以检验气味学习取决于嗅球中锌突触释放的假设。突触释放的锌通过改变可塑性而与大脑某些部分的信息存储密切相关，这是突触的活性随着刺激而随着时间的流逝而变化。奇怪的是，已知显示可塑性的嗅球中唯一的突触也具有一些最高的锌浓度。螯合剂是隔离锌的分子，将使用荧光锌指示剂来研究锌蛋白产生可塑性的作用。由于这些突触似乎参与了具有挑战性的感觉区分，因此将在学习困难的气味歧视任务后进行测试，以探测锌释放如何在嗅球中受到影响。从这些实验中收集的数据中，人们将能够确定在气味介导的学习时锌释放的模式是否变化，并得出有关锌在调节嗅觉中的重要性的结论。