Development and Application of New Optogenetic Tools Targeted to Intracellular Compartments

在第一个项目中，我们将结合质子和氯离子通道或氯离子泵与突触小泡(SV)或溶酶体的靶向动机。这一策略将基于我们最近发表的关于光驱动质子泵Arch3细胞器特异性表达对SV和溶酶体的光发生酸化的工作。为了实现来自SV和溶酶体的质子的急性耗尽，我们将使用来自Arch3的质子通道，这是Hegemann实验室最近通过点定向突变开发的。对于流明氯化物的操作，我们将实施氯离子传导通道视紫红质或光驱氯化物泵。Tetraspanin synaptophysin或CD63将分别作为SV或溶酶体的靶向序列。光驱动致动器将与遗传编码的管腔pH或膜电压的光学指示器相结合，我们将事先使用生物物理方法对其进行表征和优化。所有构建物都将在原代培养的神经细胞中表达，使用慢病毒或腺相关病毒作为病毒基因转移系统。我们新开发的工具将允许详细研究活细胞内溶酶体室内离子的动态平衡。在第二个项目中，我们将设计突触前光激活的腺苷环化酶，这将有助于光等对突触前长时程增强(LTP)的研究。我们将细菌光激活的腺苷酸环化酶(BPAC)与不同的突触前蛋白融合，并测试它们在异源表达系统中的功能，以及它们在原代神经元中的正确定位。接下来，我们将研究光激活突触前BPAC对自体培养的海马颗粒细胞突触传递的影响，我们建立了突触前LTP的体外模型。然后，我们将把突触前BPAC的应用转移到海马片培养中，并研究突触前BPAC激活对苔藓纤维传递的影响。最后，我们将通过向齿状回立体定向注射腺相关病毒颗粒在体内表达BPAC，并在急性海马片上演示其功能。光触发的突触前LTP在基因定义神经元中的诱导将为研究突触前LTP的分子机制提供新的途径。此外，它将使关于突触前LTP对学习、记忆和行为的影响的体内研究成为可能。

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.