TrkB receptor activation

描述（由申请人提供）：TrkB激活对神经元生理学的许多方面至关重要，如发育中的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有关，包括癫痫、神经性疼痛和阿尔茨海默病，这一点也不奇怪。由于TrkB在生理和病理上的重要性，阐明TrkB激活的潜在机制和由此产生的功能后果对于开发针对TrkB介导的疾病的靶向治疗至关重要。然而，不幸的是，探索这些问题受到当前技术的限制，这些技术只能提供TrkB激活的时间和空间的静态快照-通过Western Blot分析脑提取物或使用免疫组织化学分析固定组织中报告TrkB激活的磷酸化特异性抗体。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。具体来说，我假设TrkB融合到绿色荧光蛋白，PLC-delta （PLC gamma1的一个片段）融合到红色荧光蛋白将是一种有效的基于fret的传感器，专门针对Y816的TrkB激活（特定于PLC gamma1途径的激活），从而使我能够回答TrkB激活在活组织内特定神经元过程和事件中发生的地点和时间。为了验证这一假设，我将开发并优化

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. PUBLIC HEALTH RELEVANCE: The TrkB receptor is associated with many aspects of human health and disease including learning and memory, epilepsy, Alzheimer's disease, and neuropathic pain. As such, elucidating the molecular mechanisms underlying TrkB activation and the resulting functional consequences is critical for informing the design of future therapeutic strategies for TrkB- mediated diseases. This proposal seeks to develop a new research tool to aid in elucidating these mechanisms involving TrkB.