Development of light-inducible Trk receptors for neurotrophins and application

Abstract

To regulate cellular functions, many biological substances like hormones, growth factors and cytokines mediate proper cellular processes. Especially in the brain, neurotrophins are well-known to be key regulators in nervous system for the cell survival, differentiation and neuronal growth. Each neurotrophin bind to its specific Trk (tropomyosin-related kinase) receptor to mediate intracellular signaling pathways. In chapter 1, I introduce optoTrk, a new optogenetic technique to activate neurotrophin receptors with simple blue-light (488 nm) stimulation. The light-interacting module based on Arabidopsis thaliana cryptochrome 2 has homo-interacting property, used for engineering photoactivatable Trk receptors. Illumination of light efficiently activates canonical Trk downstream signaling pathways. A single light triggers transient signaling activation, reversibly reactivated with repetitive light stimuli. Moreover, the light-activated processes are tightly regulated with high spatiotemporal precision. OptoTrk system offers an effective way to control its receptor activities and experimental opportunities to study diverse biological processes both in vitro and in vivo. In chapter 2, I introduce the importance of actin waves in neuronal polarization. By activating TrkB receptor at growth cone area specifically with optoTrkB system, I found F-actin rich waves formed at somatodendritic region and moving toward the neurite tip. Light-mediated local TrkB activation at growth cone area initiates actin wave formation and following neurite elongation. Rearrangement of actin pool was emerged from local TrkB activation to cause F-actin accumulated waves and even growth cone induction at the stimulated neurite. Additionally, this local activation of TrkB receptor at growth cone brought about accumulation of developmentally regulated protein E (drebrin E) that found mostly in growing axons. Also, axon specific proteins such as End-Binding Protein 3 (EB3) and synaptophysin changed their orientation as recruited in the TrkB activated neurite specially. Thus, local TrkB activated signaling increase F-actin rich waves from altering actin balance to determine axonal fate in neuronal development.