TrkB receptor activation

描述(申请人提供)：TrkB激活对神经元生理学的许多方面至关重要，例如发育中神经元的生存和分化，以及突触的形成、功能和可塑性。由于TrkB的这些生理作用，TrkB激活的失调与包括癫痫、神经性疼痛和阿尔茨海默氏症在内的许多神经系统疾病有关也就不足为奇了。由于TrkB在生理学和病理学上的重要性，阐明TrkB激活的机制及其导致的功能后果对于开发TrkB介导的疾病的靶向治疗至关重要。然而，不幸的是，目前的技术限制了对这些问题的探索，这些技术只能提供TrkB激活的时间和空间的静态快照--通过Western Blot分析大脑提取液或使用免疫组织化学分析固定组织中报告TrkB激活的磷酸特异性抗体。为了解决这些限制并开发TrkB激活的动态读数，我建议利用荧光共振能量转移(FRET)技术和多光子成像的最新进展。具体地说，我假设融合了绿色荧光蛋白的TrkB和融合了红色荧光蛋白的PLC-Delta(PLC Gamma1的片段)将是一个有效的基于FRET的传感器，可在16年特异性激活TrkB(专用于激活PLC Gamma1途径)，从而使我能够回答在活组织内的特定神经元过程和事件中，TrkB激活发生在何时何地。为了验证这一假设，我将开发和优化 该传感器可以检测异种细胞中的TrkB受体，然后使用所开发的传感器动态记录海马齿状颗粒细胞苔藓纤维轴突中TrkB的激活情况，以响应高频刺激--据信高频刺激会激活苔藓纤维巨大胞内的TrkB。该项目的成功完成将为实时检查活组织中TrkB的激活提供一种新的工具，并有望为了解激活的潜在机制以及这种激活的功能后果--包括生理和病理--提供洞察力。

DESCRIPTION (provided by applicant): TrkB activation is critical for many aspects of neuronal physiology such as the survival and differentiation of developing neurons as well as the formation, function, and plasticity of synapses. Due to these physiologic roles, it is no surprise that dysregulation of TrkB activation has been associated with many neurologic diseases including epilepsy, neuropathic pain, and Alzheimer's. Because of TrkB's importance in both physiology and pathology, elucidating the mechanisms underlying TrkB activation and the resulting functional consequences is critical for developing targeted therapies for TrkB mediated diseases. Unfortunately though, exploring such questions has been limited by current technologies which provide only a static snapshot in time and space of TrkB activation - phospho-specific antibodies reporting TrkB activation in brain extracts analyzed by Western Blot or in fixed tissue using immunohistochemistry. To address these limitations and develop a dynamic readout of TrkB activation, I propose utilizing recent advances in fluorescence resonance energy transfer (FRET) technology and multiphoton imaging. Specifically, I hypothesize that TrkB fused to green fluorescent protein and PLC-delta (a fragment of PLC gamma1) fused to red fluorescent protein will be an effective FRET-based sensor specific for TrkB activation at Y816 (specific for activation of the PLC gamma1 pathway) and will thus enable me to answer where and when TrkB activation occurs during specific neuronal processes and events within living tissue. To test this hypothesis, I will develop and optimize the sensor in heterologous cells and then use the developed sensor to dynamically record TrkB activation within giant boutons of hippocampal dentate granule cell mossy fiber axons in response to high frequency stimulation - a stimulus believed to activate TrkB within mossy fiber giant boutons. Successful completion of this project will provide a novel tool for examining TrkB activation in real time in living tissue and promises to provide insights into mechanisms underlying activation as well as the functional consequences - both physiologic and pathologic - of such activation.