Development and Application of New Optogenetic Tools Targeted to Intracellular Compartments

点击翻译按钮获取中文摘要

The aim of our proposed work program is the development of novel optogenetic tools that allow investigations on subcellular compartments.In the first project, we will combine proton and chloride channels or chloride pumps with targeting motives for synaptic vesicles (SV) or lysosomes. The strategy will be based on our recently published work on optogenetic acidification of SV and lysosomes by organelle-specific expression of the light-driven proton pump Arch3. In order to achieve acute depletion of protons from SV and lysosomes we will use a proton channel derived from Arch3, which has been recently developed by the Hegemann lab by point-directed mutagenesis. For the manipulation of luminal chloride we will implement chloride-conductive channelrhodopsins or light-driven chloride pumps. The tetraspanin synaptophysin or CD63 will serve as targeting sequence for SV or lysosomes, respectively. The light-driven actuators will be combined with genetically encoded, optical indicators for luminal pH or membrane voltage, which we will characterize and optimize beforehand using biophysical approaches. All constructs will be expressed in primary neuronal cell cultures using lentivirus or adeno-associated virus as viral gene transfer systems. Our newly developed tools will allow detailed investigations on the ion homeostasis in the endo-lysosomal compartments in living cells. In the second project we will design presynaptic light-activated adenylyl cyclases, which will facilitate optogenetc investigations on presynaptic long-term potentiation (LTP). We will fuse the bacterial photoactivatable adenylyl cyclases (bPAC) to different presynaptic proteins and test their function in heterologous expression systems, and their correct localization in primary neurons. Next, we will study the effect of presynaptic bPAC activation by light on synaptic transmission in autaptic cultures of hippocampal granule cells, which we have established as in-vitro model for presynaptic LTP. We will then transfer the application of presynaptic bPAC to hippocampal slice cultures and investigate the impact of activation of presynaptic bPAC on mossy fiber transmission. Finally, we will express bPAC in vivo using stereotactic injections of adeno-associated viral particles into the dentate gyrus, and demonstrate its function in acute hippocampal slices. The light-triggered induction of presynaptic LTP in genetically defined neurons will allow new approaches to study the molecular mechanisms of presynaptic LTP. Further, it will enable in-vivo studies on the impact of presynaptic LTP on learning, memory and behavior.