Novel tools for spatiotemporal modulation of astrocytes in neuronal circuits

神经元回路中星形胶质细胞时空调节的新工具

• 批准号: 9810860
• 负责人: MRIGANKA SUR
• 金额: $ 154.68万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-15 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9810860
• 关键词: Ablation; Acute; Affect; Arousal; Astrocytes; Biological Process; Brain; Brain Diseases; Breeding; CRISPR/Cas technology; Calcium; Communication; Complement; Correlation Studies; Cre-LoxP; Excitatory Synapse; GTP-Binding Proteins; Gene Expression; Genes; Glutamates; Guide RNA; Image; In Situ; In Vitro; Investigation; Ion Channel Gating; Knock-out; Measures; Mediating; Membrane; Metabolic; Methodology; Methods; Modeling; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neuroglia; Neurons; Phenotype; Physiological; Physiology; Play; Preparation; Presynaptic Terminals; Process; Property; Protons; Resources; Role; Signal Transduction; Signaling Protein; Slice; Sodium; Structure; System; Tetracyclines; Time; Trans-Activators; Transgenic Mice; Transgenic Organisms; Variant; Vertebral column; Viral; Virus; adeno-associated viral vector; awake; base; calcium indicator; cost; designer receptors exclusively activated by designer drugs; experimental study; genetic makeup; imaging modality; in vivo; innovation; light gated; motor learning; mouse model; mutation screening; neural circuit; neuronal circuitry; neuronal excitability; noradrenergic; novel; novel strategies; optogenetics; postsynaptic; receptor; recombinase; response; spatiotemporal; tool; two photon microscopy; two-photon; uptake

Astrocytes are a major class of non-neuronal cells in the brain whose crosstalk with neurons at the synaptic and circuit levels remains poorly understood. While in vivo two-photon microscopy has revealed spatiotemporally diverse astrocytic signatures of intracellular Ca2+ transients, the scarcity of tools that manipulate the genetic makeup and physiological activity of astrocytes with spatial and temporal precision in vivo has restricted investigation of their physiological impact on neurons to predominantly correlational studies. Here, we propose developing three novel and mutually independent tools that target three crucial functions of astrocytes: gene expression, intracellular signal transduction, and glutamate uptake. In Aim 1, we will develop a CRISPR/Cas9- based platform to simultaneously knockout multiple genes selectively in astrocytes. Current mouse astrocytic gene ablation studies rely on a small number of Cre-LoxP recombinase transgenic lines, which target only a single gene and often lack temporal and spatial control. We propose creating a novel astrocyte-specific, temporally inducible, CRISPR/Cas9 conditional transgenic mouse model with an innovative viral platform for ablating multiple genes using a single virus Multi-gRNA, Cys4-mediated, Universal Targeting System (MRCUTS). We will apply this system in cultured astrocytes to target the Itpr2 and Adra1a/b genes (aim 1a), validate the tool and compare its efficacy to current Cre-LoxP methods (aim 1b), and probe a new functional role of astrocytes in arousal by using MRCUTS to simultaneously ablate two subtypes of noradrenergic receptors (Adra1a/b) (aim 1c). In Aim 2, we will develop a method for optogenetically activating G-protein signaling cascades in astrocytes. Current methods for modulating astrocyte signaling, such as DREADDs, lack temporal precision. We will develop and characterize the use of optogenetically activated G-protein receptors (opto-XR) in astrocytes to probe astrocyte signal transduction on physiologically-relevant timescales, first in vitro (aim 2a), then in vivo using 2-photon microscopy to measure astrocyte calcium dynamics (aim 2b), and subsequently explore the effects of astrocytic G-protein signal transduction on neuronal physiology using opto-XR in conjunction with astrocyte-neuron dual-calcium imaging (aim 2c). In Aim 3, we will develop an in vivo method for optogenetically disrupting glutamate uptake by astrocytes. Screening for mutations in ChR2, and combining four mutations, results in a light-gated ion channel, ChromeQ that possesses order-of-magnitude reductions in calcium and proton conductance while increasing sodium currents. We will record from astrocytes in acute brain slices to parameterize optogenetically activated sodium currents and determine effects on both astrocyte transporter currents and nearby neurons (aim 3a), examine how disrupting glutamate uptake via chromeQ affects astrocyte calcium dynamics and neuronal response properties in vivo (aim 3b), and explore the effects of ChromeQ on neuronal physiology and motor learning (aim 3c). The tools proposed here will enable a deeper understanding of astrocyte-neuron crosstalk in normal brain function and its disruption in brain disorders.

星形胶质细胞是大脑中的一类主要的非神经元细胞，其在突触处与神经元进行串扰， 和电路水平仍然知之甚少。虽然体内双光子显微镜已经揭示了时空 细胞内Ca 2+瞬变的星形胶质细胞特征多样性，操纵基因表达的工具的缺乏， 星形胶质细胞的组成和生理活性在体内的空间和时间精度受到限制， 研究其对神经元的生理影响，主要是相关性研究。在此，我们建议 开发三种新的、相互独立的工具，靶向星形胶质细胞的三种关键功能：基因 表达、细胞内信号转导和谷氨酸摄取。在目标1中，我们将开发CRISPR/Cas9- 在星形胶质细胞中同时选择性地敲除多个基因。当前小鼠星形胶质细胞 基因消除研究依赖于少量的Cre-LoxP重组酶转基因系，其仅靶向一个 单基因，往往缺乏时间和空间控制。我们建议创造一种新的星形胶质细胞特异性的， 时间可诱导的CRISPR/Cas9条件转基因小鼠模型，具有创新的病毒平台， 使用单一病毒消除多个基因多gRNA，Cys 4介导的，通用靶向系统 （MRCUTS）。我们将在培养的星形胶质细胞中应用该系统以靶向Itpr 2和Adra 1a/B基因（目的1a）， 验证该工具，并将其有效性与当前的Cre-LoxP方法进行比较（目的1b），并探索新的功能作用 通过使用MRCUTS同时消融两种亚型的去甲肾上腺素能受体， （Adra 1a/B）（目标1c）。在目标2中，我们将开发一种光遗传学激活G蛋白信号传导的方法 星形胶质细胞的级联反应目前用于调节星形胶质细胞信号传导的方法，如DREADD，缺乏时间上的限制。 精度我们将开发和表征使用光遗传学激活的G蛋白受体（opto-XR） 在星形胶质细胞中以生理相关时间尺度探测星形胶质细胞信号转导，首先在体外（AIM 2A）， 然后在体内使用双光子显微镜测量星形胶质细胞钙动力学（aim 2b），随后 利用opto-XR技术研究星形胶质细胞G蛋白信号转导对神经元生理的影响， 结合星形胶质细胞-神经元双钙成像（AIM 2C）。在目标3中，我们将开发一种体内方法， 光遗传学干扰星形胶质细胞摄取谷氨酸。筛选ChR 2的突变，并结合四种 突变，导致光门控离子通道，ChromeQ，其具有数量级的减少， 钙和质子电导，同时增加钠电流。我们将从急性脑损伤的星形胶质细胞中记录 切片以参数化光遗传学激活的钠电流，并确定对星形胶质细胞 转运体电流和附近的神经元（目的3a），研究如何通过chromeQ破坏谷氨酸摄取影响 星形胶质细胞钙动力学和神经元反应特性在体内（目的3b），并探讨影响 ChromeQ对神经生理学和运动学习的影响（aim 3c）。这里提出的工具将使更深入的 了解正常脑功能中星形胶质细胞-神经元串扰及其在脑疾病中的破坏。