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: 9187451
• 负责人: Yan Qin
• 金额: $ 21.28万
• 依托单位: UNIVERSITY OF DENVER (COLORADO SEMINARY)
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9187451
• 关键词: Address; Affect; Affinity; Alpha Cell; Animals; Binding; Biological; Biophysics; 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; Light; Location; Maintenance; Measures; Memory; Mentors; Methodology; Methods; Modeling; Molecular Conformation; Monitor; Mutagenesis; Mutate; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Noise; Organism; Pathologic; Phase; Physiological; Positioning Attribute; Proteins; Protons; Recovery; Recycling; Regulation; Reperfusion Therapy; Reporting; Research; Role; SLC30A3 gene; Signal Transduction; Synapses; Synaptic Vesicles; Technology; Testing; Time; Transfection; Transgenic Organisms; Vesicle; Work; Zebrafish; Zinc Fingers; antiporter; base; biophysical properties; design; experimental study; fluorescence imaging; imaging system; neuroprotection; neurotransmission; novel; protein structure; prototype; public health relevance; response; screening; sensor; small molecule; spatiotemporal; 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（申请人提供）：突触小泡 Zn2+ 被认为是一种神经信号调节剂，因此我的长期目标是研究小泡 Zn2+ 如何调节脑功能，并确定突触小泡 Zn2+ 稳态失衡参与神经退行性变和脑损伤的机制。监测神经元突触小泡 Zn2+ 对于实现这一目标至关重要。目前，小分子 Zn2+ 传感器用于可视化囊泡 Zn2+；然而，小分子传感器因其非特异性定位和无法进行长期成像而受到限制。我提出的研究的主要目标是生成生物成像系统，能够以高时空保真度监测活体神经元和动物中的囊泡 Zn2+ 动态。所提出的生物成像系统正在利用基因编码传感器的能力进行特定靶向（特殊细胞群中的特定亚细胞位置）和长期成像。将开发一种基于基因编码的新型单荧光蛋白（single-FP）的 Zn2+ 传感器，并将其靶向神经元的突触小泡。在初步研究中，原型单FP Zn2+传感器是通过将转录因子Zap1（ZF1和ZF2）的两个锌指连接到循环排列荧光蛋白（FP）的两端而产生的。当Zn2+结合时，形成两个锌指折叠，引起指-指相互作用，从而引起FP随后的构象变化和荧光强度的变化。在拟议研究的指导阶段，将使用基于细胞的突变传感器库筛选来优化原型单FP传感器以获得更好的荧光信号。经过验证的单 FP Zn2+ 传感器将被整合到突触囊泡中，然后将其引入培养的神经元和斑马鱼中，生成基于细胞和动物的成像系统。在独立阶段，两个成像系统都将被评估并应用于生物学研究。我将测试一个具体的假设：突触囊泡 Zn2+ 转运蛋白 ZnT3 在缺血/再灌注期间利用 Zn2+/质子交换机制将 Zn2+ 浓缩到囊泡中。此外，我还将探讨突触小泡Zn2+在斑马鱼缺血性脑损伤中的作用。这些研究不仅将验证成像系统的实用性，还将揭示突触小泡Zn2+的调节机制及其对完整活体动物脑缺血/再灌注期间神经元恢复的具体影响。总之，本研究将开发新的成像工具，以高空间和时间保真度监测活体神经元和动物中的突触小泡 Zn2+，这将为研究突触小泡 Zn2+ 的信号功能提供新方法。此外，在缺血模型中使用这些成像系统可以阐明如何调节突触囊泡 Zn2+ 以实现缺血/再灌注期间神经元的恢复。