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

描述（申请人提供）：突触水疱性Zn2+被认为是一种神经元信号调节剂，因此我的长期目标是研究水疱性Zn2+如何调节脑功能，并确定突触水疱性Zn2+失衡参与神经变性和脑损伤的机制。监测神经元突触囊泡Zn2+对实现这一目标具有重要意义。目前，小分子Zn2+传感器用于可视化水疱状Zn2+；然而，小分子传感器受限于其非特异性定位和无法进行长期成像。我提出的研究的主要目标是生成生物成像系统，可以监测活神经元和动物中具有高时空保真度的囊泡Zn2+动态。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（特定细胞群中的特定亚细胞位置）和长期成像。一种基于基因编码Zn2+传感器的新型单荧光蛋白（single- fp）将被开发并靶向到神经元突触囊泡中。在初步研究中，通过将转录因子Zap1的两个锌指（ZF1和ZF2）连接到环状排列荧光蛋白（FP）的两端，生成了单FP Zn2+传感器的原型。当Zn2+结合时，形成两个锌指褶，引起手指-手指相互作用，从而引起FP的后续构象变化和荧光强度的变化。在建议研究的指导阶段，原型单fp传感器将通过基于细胞的突变传感器库筛选来优化更好的荧光信号。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，这两种成像系统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+转运体ZnT3利用Zn2+/质子交换机制在缺血/再灌注时将Zn2+浓缩到囊泡中。此外，我将探讨突触囊泡Zn2+在斑马鱼缺血性脑损伤中的作用。这些研究不仅可以验证成像系统的实用性，还可以揭示突触囊泡Zn2+的调控机制及其对完整活体脑缺血再灌注时神经元恢复的特异性作用。综上所述，本研究将为活体神经元和动物突触囊泡Zn2+的监测提供新的成像工具，具有较高的时空保真度，为研究突触囊泡Zn2+的信号功能提供新的方法。此外，利用这些成像系统在缺血模型中可以阐明如何调节突触囊泡Zn2+在缺血/再灌注期间促进神经元恢复。

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.