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

工程新的光遗传学蛋白质和生物传感器：研究神经胶质信号传导的工具

• 批准号: RGPIN-2019-06274
• 负责人: Lohman, Alexander
• 金额: $ 2.19万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=738813
• 关键词: Engineering; new; optogenetic; proteins; biosensors

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.

越来越多的人认识到，神经胶质细胞，大脑中的非神经元细胞，动态调节单个突触和神经元网络功能的方式。神经胶质在调节突触形成和消除、突触可塑性以及神经元死亡和功能障碍方面发挥着双重作用。在中枢神经系统中，胶质细胞、小胶质细胞和星形胶质细胞合成并释放多种信号分子，动态调节突触传递和神经回路动力学，包括促炎和抗炎细胞因子、趋化因子和神经营养因子。虽然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自基因组编辑技术出现以来已经扩大，但在将神经胶质信号传导的时空信息与神经元功能动力学相关联方面仍然存在空白。这主要是由于在测量局部浓度和时间动态的个别信号分子和操作的异质性胶质细胞群体在完整的大脑中的特定方式的限制。光遗传学，即光控蛋白质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。基因编码的钙指示剂和电压传感器与光学控制的离子通道和泵的组合现在有助于直接测量神经元活动，能够激发或沉默个体或群体的神经元，对大脑组织和各种认知，运动和感觉功能的编码提供无与伦比的光线。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传导的理解，但目前缺乏用于神经胶质研究的互补光控蛋白和生物传感器。通过用新的以胶质细胞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胶质细胞信号动力学以及对神经元信号和电路动力学的影响的理解。这个发现补助金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）创建新的遗传编码的光可释放信号分子; 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质以操纵神经胶质表型和效应器功能。该提案特别关注炎症细胞因子白细胞介素-1 β（IL-1b），因为它在神经胶质信号传导中起着重要作用。利用我们新的光可切割蛋白质技术，我们将产生一种光学控制的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种新的IL-1b生物传感器，并设计一种新型光学控制的NFkB转录因子系统来调节IL-1b信号下游的基因表达。