Development and Application of New Optogenetic Tools Targeted to Intracellular Compartments

我们提出的工作计划的目的是开发新的光遗传学工具，使调查亚细胞compartments.In第一个项目中，我们将结合联合收割机质子和氯离子通道或氯离子泵与突触囊泡（SV）或溶酶体的靶向动机。该策略将基于我们最近发表的关于SV和溶酶体通过光驱动质子泵Arch3的细胞器特异性表达的光遗传学酸化的工作。为了实现SV和溶酶体质子的急性消耗，我们将使用来自Arch3的质子通道，其最近由Hegemann实验室通过定点诱变开发。对于管腔氯的操纵，我们将实施氯传导通道视紫红质或光驱动氯泵。四跨膜蛋白突触体蛋白或CD 63将分别用作SV或溶酶体的靶向序列。光驱动致动器将与基因编码的光学指示剂结合，用于管腔pH值或膜电压，我们将使用生物物理方法预先表征和优化。所有构建体将使用慢病毒或腺相关病毒作为病毒基因转移系统在原代神经元细胞培养物中表达。我们新开发的工具将允许在活细胞内的内溶酶体隔间的离子稳态的详细调查。在第二个项目中，我们将设计突触前光激活的腺苷酸环化酶，这将有助于光基因研究突触前长时程增强（LTP）。我们将细菌的光活化腺苷酸环化酶（bPAC）融合到不同的突触前蛋白，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，我们将研究突触前bPAC激活光对突触传递的海马颗粒细胞，我们已经建立了突触前LTP的体外模型的自适应培养的影响。然后，我们将转移应用突触前bPAC海马切片文化和研究的影响，激活突触前bPAC苔藓纤维传输。最后，我们将表达bPAC在体内使用立体定向注射腺相关病毒颗粒到齿状回，并证明其在急性海马切片的功能。在遗传上确定的神经元中光触发的突触前LTP的诱导将允许新的方法来研究突触前LTP的分子机制。此外，它将使突触前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.