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.
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.
| Hedrick, Nathan G; Harward, Stephen C; Hall, Charles E et al. (2016) Rho GTPase complementation underlies BDNF-dependent homo- and heterosynaptic plasticity. Nature 538:104-108 |
| Harward, Stephen C; Hedrick, Nathan G; Hall, Charles E et al. (2016) Autocrine BDNF-TrkB signalling within a single dendritic spine. Nature 538:99-103 |
| Harward, Stephen C; McNamara, James O (2014) Aligning animal models with clinical epilepsy: where to begin? Adv Exp Med Biol 813:243-51 |
| Harward, Stephen C; McNamara, James O (2013) In search of the ever-elusive positive endozepine. Neuron 78:951-2 |
