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

描述（申请人提供）：突触囊泡Zn 2+被认为是神经元信号传导调节剂，因此我的长期目标是研究囊泡Zn 2+如何调节脑功能，并确定突触囊泡Zn 2+稳态异常参与神经变性和脑损伤的机制。监测神经元突触囊泡中的Zn 2+对于实现这一目标具有重要意义。目前，小分子Zn 2+传感器被用于可视化囊泡Zn 2 +;然而，小分子传感器受到其非特异性定位和无法长期成像的限制。我提出的研究的主要目标是产生生物成像系统，可以监测囊泡Zn 2+动态活神经元和动物具有较高的时空保真度。拟议的生物成像系统正在利用基因编码传感器的能力，用于特定靶向（特定细胞群中的特定亚细胞位置）和长期成像。一种新的单荧光蛋白（单FP）为基础的基因编码的Zn 2+传感器将被开发和靶向神经元突触囊泡。在初步研究中，通过将转录因子Zap 1的两个锌指（ZF 1和ZF 2）连接到环形排列的荧光蛋白（FP）的两端来产生原型单FP Zn 2+传感器。当Zn ~（2+）被结合时，两个锌指折叠的形成会引起指-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。在所提出的研究的指导阶段，将使用突变传感器文库的基于细胞的筛选来优化原型单FP传感器以获得更好的荧光信号。然后将验证的单FP Zn 2+传感器并入突触囊泡中，然后将其引入培养的神经元和斑马鱼中，产生基于细胞和基于动物的成像系统。在独立阶段，将对两种成像系统进行评估并应用于生物学研究。我将测试一个具体的假设：突触囊泡锌+转运蛋白ZnT 3利用锌+/质子交换机制，集中锌2+进入囊泡在缺血/再灌注。此外，我将探索突触囊泡Zn 2+在斑马鱼缺血性脑损伤中的作用。这些研究不仅验证了该成像系统的实用性，而且揭示了突触囊泡Zn ~（2+）的调控机制及其在完整活体动物脑缺血/再灌注神经元恢复中的特异性作用。总之，该研究将开发新的成像工具，用于在活体神经元和动物中监测突触囊泡Zn 2+，具有高度的空间和时间保真度，这将为研究突触囊泡Zn 2+的信号功能提供新的方法。此外，在缺血模型中利用这些成像系统可以阐明如何调节突触囊泡Zn 2+用于缺血/再灌注期间的神经元恢复。

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.