Generation of Cell-based and Animal-based Imaging Systems for Monitoring Synaptic

生成用于监测突触的基于细胞和动物的成像系统

• 批准号: 8764915
• 负责人: Yan Qin
• 金额: $ 8.94万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8764915
• 关键词: Address; Affect; Affinity; Animals; Binding; Biological; Brain; Brain Injuries; Brain Ischemia; Cells; DNA Sequence; Development; Environment; Family; Fingers; Fluorescence; Generations; Glutamates; Goals; Health; Homeostasis; Human; Image; Imaging Device; In Vitro; Injury; Investigation; Ions; Ischemia; Ischemic Brain Injury; Ischemic Neuronal Injury; Learning; Libraries; Life; Light; Location; Maintenance; Measures; Memory; Mentors; Methods; Modeling; Monitor; Mutagenesis; Mutate; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Noise; Organism; Phase; Physiological; Positioning Attribute; Proteins; Protons; Recovery; Recycling; Regulation; Reperfusion Therapy; Reporting; Research; Role; Signal Transduction; Staging; Synapses; Synaptic Vesicles; System; Technology; Testing; Time; Transfection; Transgenic Organisms; Vesicle; Work; Zebrafish; Zinc Fingers; antiporter; base; biophysical properties; design; fluorescence imaging; neuroprotection; novel; protein structure; prototype; public health relevance; research study; response; screening; sensor; small molecule; synaptic function; tool; transcription factor; vector

DESCRIPTION (provided by applicant): Synaptic vesicular Zn2+ has been regarded as a neuronal signaling modulator, thus my long-term goal is to study how the vesicular Zn2+ regulates brain function and to identify the mechanism by which synaptic vesicular Zn2+ dyshomeostasis is involved in neurodegeneration and brain injury. Monitoring the synaptic vesicular Zn2+ in neurons is of critical significance for achieving this goal. Currently, small molecule Zn2+ sensors were used to visualize vesicular Zn2+; however the small molecule sensors are limited by their nonspecific localizations and inability for long-term imaging. The major objectives of my proposed research are to generate biological imaging systems that can monitor vesicular Zn2+ dynamics in living neurons and animals with high spatio-temporal fidelity. The proposed biological imaging systems are exploiting the capability of genetically encoded sensors for specific targeting (specific subcelluar locations in specialized groups of cells) and long-term imaging. A novel single fluorescent protein (single-FP) based genetically encoded Zn2+ sensors will be developed and targeted into synaptic vesicles in neurons. In the preliminary studies, the prototype single-FP Zn2+ sensors were generated by attaching two zinc fingers of transcription factor Zap1 (ZF1 and ZF2) to the two ends of circularly permuted fluorescent protein (FP). When Zn2+ is bound, the formation of two zinc finger folds would cause the finger-finger interaction, which would induce subsequent conformational change of FP and the changes of fluorescent intensities. In the mentored phase of proposed research, the prototype single-FP sensors will be optimized for better fluorescent signals using cell- based screening of mutated sensor library. The validated single-FP Zn2+ sensors will then be incorporated into the synaptic vesicles, which will then be introduced into cultured neurons and zebrafish, generating cell-based and animal-based imaging systems. In the independent phase, both imaging systems will be evaluated and applied to biological studies. I will test a specific hypothesis: synaptic vesicular Zn2+ transporter ZnT3 utilizes a Zn2+/proton exchange mechanism to concentrate Zn2+ into vesicles during ischemia/reperfusion. In addition, I will explore the roles of synaptic vesicular Zn2+ in ischemic brain damage in zebrafish. These studies would not only verify the practicability of the imaging systems, but also discover the regulation mechanism of synaptic vesicular Zn2+ and their specific effects on neuronal recovery during brain ischemia/reperfusion in intact living animals. In conclusion, the proposed research will develop new imaging tools for monitoring synaptic vesicular Zn2+ in living neurons and animals with high spatial and temporal fidelity, which will offer a new method to study the signaling function of synaptic vesicular Zn2+. Additionally, utilization of these imaging systems in the ischemia models could elucidate how to modulate the synaptic vesicular Zn2+ for neuronal recovery during ischemia/reperfusion.

描述（由申请人提供）：突触囊泡Zn2+已被视为神经元信号调节剂，因此我的长期目标是研究如何调节大脑功能的囊泡Zn2+如何识别突触囊泡囊泡Zn2+双性恋性疾病的机制，与神经降解和脑损伤有关。监测神经元中的突触囊泡Zn2+对于实现这一目标至关重要。当前，小分子Zn2+传感器用于可视化囊泡Zn2+。但是，小分子传感器受其非特异性局部化和长期成像的限制。我拟议的研究的主要目标是生成生物成像系统，该系统可以监测具有较高时空保真度的活神经元和动物中的囊泡Zn2+动力学。所提出的生物成像系统正在利用遗传编码的传感器对特定靶向的能力（特定的细胞组中的特定亚核位置）和长期成像。将开发出一种新型的基于遗传编码的Zn2+传感器的单个荧光蛋白（单FP），并将其靶向神经元中的突触囊泡。在初步研究中，通过将两个转录因子ZAP1（ZF1和ZF2）的锌指Zn2+传感器连接到圆形置换荧光蛋白（FP）的两端来产生。当Zn2+结合时，两个锌指折叠的形成会导致指手指相互作用，这会引起FP的随后构象变化和荧光强度的变化。在拟议的研究的指导阶段，将使用突变传感器库的基于细胞基的筛选来优化原型单FP传感器，以更好地使用荧光信号。然后将经过验证的单FP ZN2+传感器掺入突触囊泡中，然后将其引入培养的神经元和斑马鱼中，从而产生基于细胞的和基于动物的成像系统。在独立阶段，两个成像系统将被评估并应用于生物学研究。我将检验一个特定的假设：突触囊泡Zn2+转运蛋白Znt3利用Zn2+/质子交换机制将Zn2+浓缩到缺血/再灌注期间。此外，我将探讨突触囊泡Zn2+在斑马鱼中缺血性脑损伤中的作用。这些研究不仅会验证成像系统的实用性，而且还会发现突触囊泡Zn2+的调节机制及其对完整活动物中脑缺血/再灌注过程中神经元恢复的特定影响。总之，拟议的研究将开发新的成像工具，用于监测具有高空间和时间忠诚度的活神经元和动物的突触囊泡Zn2+，这将提供一种新的方法来研究突触囊泡Zn2+的信号传导功能。此外，缺血模型中这些成像系统的利用可以阐明如何调节缺血/再灌注过程中神经元恢复的突触囊泡Zn2+。