TrkB receptor activation

描述（由申请人提供）： TrkB激活对于神经元生理学的许多方面是至关重要的，例如发育神经元的存活和分化以及突触的形成、功能和可塑性。由于这些生理作用，TrkB激活的失调与许多神经系统疾病包括癫痫、神经性疼痛和阿尔茨海默氏症相关也就不足为奇了。由于TrkB在生理学和病理学中的重要性，阐明TrkB活化的机制和所产生的功能后果对于开发TrkB介导的疾病的靶向疗法至关重要。然而不幸的是，探索这些问题受到当前技术的限制，这些技术仅提供TrkB活化的时间和空间上的静态快照-磷酸特异性抗体报告通过蛋白质印迹分析的脑提取物中或使用免疫组织化学分析的固定组织中的TrkB活化。为了解决这些限制，并开发一个动态读出TrkB激活，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。具体来说，我假设TrkB融合到绿色荧光蛋白和PLC-δ（PLC γ 1的片段）融合到红色荧光蛋白将是一个有效的基于FRET的传感器特异性TrkB激活在Y816（特异性激活PLC γ 1通路），从而使我能够回答何时何地TrkB激活发生在特定的神经元过程和活组织内的事件。为了验证这一假设，我将开发和优化 传感器在异源细胞中，然后使用开发的传感器动态记录TrkB激活海马齿状颗粒细胞苔藓纤维轴突的巨结内响应于高频刺激-刺激被认为激活苔藓纤维巨结内的TrkB。该项目的成功完成将提供一种新的工具，用于检查TrkB激活在活组织中的真实的时间，并承诺提供深入了解激活的机制以及功能后果-生理和病理-这样的激活。

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.