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

描述(申请人提供)：突触囊泡锌离子一直被认为是一种神经元信号调节器，因此我的长期目标是研究突触囊泡锌离子如何调节脑功能，并确定突触囊泡锌离子代谢紊乱参与神经退行性变和脑损伤的机制。监测神经元中突触囊泡的锌离子对于实现这一目标具有重要意义。目前，小分子锌离子传感器主要用于囊泡状锌离子的可视化，但由于其非特异性的局域性和不能进行长期成像而受到限制。我提出的研究的主要目标是建立生物成像系统，能够以高时空保真度监测活的神经元和动物中的锌离子泡动力学。拟议的生物成像系统正在利用遗传编码传感器的能力进行特定的靶向(特殊细胞组中的特定亚细胞位置)和长期成像。一种新的基于单一荧光蛋白(Single-FP)的基因编码的锌离子传感器将被开发出来，并靶向神经元中的突触小泡。在初步研究中，将转录因子Zap1的两个锌指(ZF1和ZF2)连接到环状排列的荧光蛋白(FP)的两端，形成了单FP锌离子传感器的原型。当锌离子结合时，两个锌指褶皱的形成会引起指-指相互作用，从而引起FP的构象变化和荧光强度的变化。在拟议研究的指导阶段，原型单FP传感器将通过基于细胞的突变传感器文库筛选来优化以获得更好的荧光信号。经过验证的单FP锌离子传感器将被整合到突触小泡中，然后被引入培养的神经元和斑马鱼中，产生基于细胞和动物的成像系统。在独立阶段，将对两种成像系统进行评估并将其应用于生物学研究。我将检验一个特定的假设：在缺血/再灌流过程中，突触囊泡锌离子转运体ZnT3利用锌离子/质子交换机制将锌离子浓缩到囊泡中。此外，我还将探讨突触囊泡锌离子在斑马鱼缺血性脑损伤中的作用。这些研究不仅验证了成像系统的实用性，还将揭示在活体动物脑缺血/再灌注过程中突触囊泡锌离子的调节机制及其对神经元恢复的特异性影响。综上所述，本研究将为高时空保真度监测活体神经元和动物突触囊泡锌离子提供新的成像手段，为研究突触囊泡锌离子的信号功能提供一种新的方法。此外，在缺血模型中利用这些成像系统可以阐明如何在缺血/再灌流期间调节突触囊泡锌离子以促进神经元的恢复。

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.