Engineering new optogenetic proteins and biosensors: Tools to study glial signaling

神经胶质细胞是大脑中的非神经元细胞，它动态地调节着单个突触和神经元网络的功能，这一点越来越受到人们的重视。胶质细胞在调节突触形成和消除、突触可塑性和神经元死亡和功能障碍方面发挥着双重作用。中枢神经系统的胶质细胞、小胶质细胞和星形胶质细胞合成并释放多种动态调节突触传递和神经回路动力学的信号分子，包括促炎性和抗炎性细胞因子、趋化因子和神经营养因子。尽管自基因组编辑技术出现以来，我们对这些信号分子在调节大脑发育、突触可塑性和神经元功能障碍方面的重要性的认识已经扩大，但在将胶质信号的时空信息与神经元功能动力学联系起来方面仍然存在空白。这主要是由于测量单个信号分子的局部浓度和时间动态以及在完整大脑中以特定方式操纵异质胶质群体的局限性。光遗传学是光控制蛋白和生物传感器的遗传编码，对完整大脑中神经元信号动力学的研究产生了巨大的影响。基因编码的钙指示器和电压传感器与光控离子通道和泵的结合，现在促进了神经元活动的直接测量，具有激活或沉默单个或神经元群体的能力，为大脑组织和各种认知、运动和感觉功能的编码提供了无与伦比的启示。虽然这些最先进的工具已经显著影响了我们对神经元信号的理解，但目前还缺乏互补的光控蛋白和生物传感器来研究胶质细胞。通过用新的以神经胶质为中心的光遗传学工具扩展实验工具箱，我们将大大增加我们对神经胶质信号动力学的理解以及对神经元信号和电路动力学的影响。这项发现资助申请旨在通过关注三个中心轴来满足这些需求。我们的具体目标是：1)创造新的遗传编码的光释放信号分子；2)为神经胶质衍生的信号分子设计遗传编码的生物传感器；3)设计新的光遗传蛋白来操纵神经胶质表型和效应功能。由于炎症细胞因子白细胞介素-1 β (IL-1b)在神经胶质信号传导中起着核心作用，因此本建议特别关注它。利用我们新的光可切割蛋白技术，我们将生成一个光控蛋白酶系统来处理和释放成熟的IL-1b，利用基于fret的技术和IL-1b受体复合物的结构信息来创建一个新的IL-1b生物传感器，并设计一个新的光控NFkB转录因子系统来调节IL-1b信号传导下游的基因表达。

It is increasingly appreciated that glia, the non-neuronal cells in the brain, dynamically regulate the way individual synapses and neuronal networks function. Glia perform dichotomous roles in regulating synapse formation and elimination, synaptic plasticity, and neuronal death and dysfunction. Of central nervous system glia, microglia and astrocytes synthesize and release a variety of signaling molecules that dynamically regulate synaptic transmission and neural circuit dynamics, including pro- and anti-inflammatory cytokines, chemokines and neurotrophic factors. While our knowledge surrounding the importance of these signaling molecules in regulating brain development, synaptic plasticity and neuronal dysfunction has expanded since the advent of genome editing technologies, there remains a void in correlating spatiotemporal information on glial signaling with neuronal functional dynamics. This is mainly due to limitations in measuring local concentrations and temporal dynamics of individual signaling molecules and manipulation of heterogeneous glial populations in specific ways in the intact brain. Optogenetics, the genetic encoding of optically-controlled proteins and biosensors, has had a tremendous impact on the study of neuronal signaling dynamics in the intact brain. The combination of genetically encoded calcium indicators and voltage sensors with optically-controlled ion channels and pumps now facilitates direct measurements of neuronal activity with the ability to excite or silence individual or populations of neurons, shedding unparalleled light on brain organization and the encoding of diverse cognitive, locomoter and sensory functions. While these state-of-the-art tools have significantly impacted our understanding of neuronal signaling, complementary light-controlled proteins and biosensors are currently lacking for the study of glia. By expanding the experimental toolbox with novel glia-centric optogenetic tools, we will considerably increase our understanding of glial signaling dynamics and the impact on neuronal signaling and circuit dynamics. This Discovery Grant application aims to meet these needs by focusing on three central axes. We specifically aim to 1) create new genetically-encoded photoreleasable signaling molecules 2) engineer genetically-encoded biosensors for glial derived signaling molecules and 3) engineer new optogenetic proteins to manipulate glial phenotypes and effector functions. This proposal specifically focuses on the inflammatory cytokine interleukin-1 beta (IL-1b) due to its central role in glial signaling. Utilizing our new photocleavable protein technology we will generate an optically-controlled protease system to process and release mature IL-1b, utilize FRET-based technology and structural information for the IL-1b receptor complex to create a new IL-1b biosensor and engineer a novel optically-controlled NFkB transcription factor system to regulate gene expression downstream of IL-1b signaling.