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

基本信息

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
项目类别：
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

项目摘要

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主要的非神经元细胞，其在突触处与神经元的串扰电路水平仍然很少了解。虽然体内两光子显微镜揭示了时空细胞内Ca2+瞬变的各种星形胶质细胞特征，操纵遗传的工具的稀缺性星形胶质细胞具有空间和时间精度的体内的化妆和生理活性已限制研究其生理对神经元主要相关研究的影响。在这里，我们建议开发三种针对星形胶质细胞三个关键功能的新型和相互独立的工具：基因表达，细胞内信号转导和谷氨酸摄取。在AIM 1中，我们将开发CRISPR/CAS9- 基于在星形胶质细胞中选择性敲除多个基因的基于平台。当前的小鼠星形细胞基因消融研究依赖于少数CRE-LoxP重组酶转基因系，仅针对A 单个基因，通常缺乏时间和空间控制。我们建议创建一种新颖的星形胶质细胞特异性，具有创新病毒平台的时间诱导，CRISPR/CAS9条件转基因小鼠模型使用单个病毒多gRNA，CYS4介导的通用靶向系统烧毁多个基因（MRCUTS）。我们将在培养的星形胶质细胞中应用该系统以靶向ITPR2和ADRA1A/B基因（AIM 1A），，验证该工具并将其功效与当前的Cre-loxp方法（AIM 1B）进行比较，并探测新的功能作用通过使用MRCUTS同时烧毁甲肾上腺素能受体的两个亚型，在唤醒中的星形胶质细胞（ADRA1A/B）（AIM 1C）。在AIM 2中，我们将开发一种用于激活G蛋白信号传导的方法星形胶质细胞中的级联反应。当前调节星形胶质细胞信号传导的方法，例如Dreadds，缺乏时间精确。我们将开发并表征使用光遗传学活化的G蛋白受体（Opto-XR）在星形胶质细胞中以探测与生理相关的时间表的星形胶质细胞信号转导，首先是体外（AIM 2A），然后使用2光子显微镜在体内测量星形胶质细胞钙动力学（AIM 2B），然后随后探索星形细胞G蛋白信号转导的影响对使用Opto-XR In In In In In In In In In In In In In In In In opto-XR 与星形胶质细胞神经元的双钙成像（AIM 2C）的结合。在AIM 3中，我们将开发一种体内方法在光遗传学上破坏了星形胶质细胞的谷氨酸吸收。筛选CHR2中的突变，并组合四个突变，导致光门控的离子通道，Chromeq具有降低的速度顺序钙和质子电导，同时增加钠电流。我们将从急性大脑中的星形胶质细胞中记录切片以参数化光遗传学活化的钠电流，并确定对两个星形胶质细胞的影响转运蛋白电流和附近的神经元（AIM 3A），检查通过Chromeq造成谷氨酸摄取的影响如何影响体内星形胶质细胞钙动力学和神经元反应特性（AIM 3B），并探索 Chromeq关于神经元生理学和运动学习（AIM 3C）。这里提出的工具将使更深入了解正常脑功能中星形胶质细胞 - 神经元串扰及其在脑部疾病中的破坏。