Protein ticker-tapes for brain-wide neural recordings

用于全脑神经记录的蛋白质自动收报机磁带

• 批准号: 10598626
• 负责人: Adam Ezra Cohen
• 金额: $ 33.8万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10598626
• 关键词: Behavior; Brain; Bypass; Capsid; Cells; Chimeric Proteins; Climate; Color; Darkness; Data; Disease; Dyes; Electrodes; Engineering; Event; Fiber; Filament; Gene Activation; Gene Expression; Gene Proteins; Goals; Growth; Health; Image; Immediate-Early Genes; In Vitro; Label; Maps; Measurement; Measures; Molecular; Nervous System; Neurons; Optics; Outcome; Peptide Hydrolases; Peptides; Physical Chemistry; Physiological; Positioning Attribute; Process; Protein Engineering; Protein Subunits; Proteins; Proteolysis; Protocols documentation; Recording of previous events; Reporter Genes; Research; Resolution; Sampling; Structure; TEV protease; Time; Tissues; Trees; Viral Vector; Work; base; biophysical chemistry; brain cell; ex vivo imaging; follow-up; high resolution imaging; in vivo; inducible Cre; inducible gene expression; inhibitor; insight; mathematical model; mechanical properties; neural; neural patterning; neuroimaging; novel strategies; portability; promoter; receptor; success; tool

PROJECT SUMMARY/ABSTRACT Protein ticker-tapes for brain-wide neural recordings Behavior emerges from the interacting activity of widely distributed ensembles of neurons; but all existing tools for measuring brain activity sample only a small subset of these dynamics. Here we propose a protein-based approach to record brain-wide dynamics of two key measures of neural activity: immediate early gene (IEG) expression and Ca2+ concentration. This proposal focuses on in vitro proof of concept; follow-up efforts will focus on in vivo application if warranted. Tree rings and ticker tapes. A core taken in a tree today can reveal climate events from centuries past. The present proposal adapts this idea to record the history of neural activation. The idea is to form slowly growing intracellular protein filaments whose position-dependent color reflects the history of IEG activity or Ca2+ dynamics. High-resolution imaging ex vivo reads this history. The steps to achieve this goal are: 1) Engineer protein fibers as molecular ticker-tapes. The HaloTag receptor (HT) will be fused to filament-forming proteins (FFPs) that form linearly growing structures in cells. Sequential addition of different- colored brain-permeant HaloTag dyes will create colored stripes in the growing filaments, mapping fiber growth to wall-clock time. Protein engineering will be used to optimize the nucleation, growth, labeling and mechanical properties of these fibers. 2) Recording IEG activity on protein fibers. A HT-FFP gene will be expressed under a constitutive promoter and eGFP-FFP under an IEG promoter. IEG activation will lead to green stripes in the fiber, whose timing will be determined by reference to the different-colored HaloTag stripes. Promoters and protein stability will be optimized to achieve high-resolution and long time-base IEG recordings. 3) Record Ca2+ dynamics on protein fibers. The HT receptor will be engineered to contain a TEV protease recognition motif (termed HT*) which inactivates the HT when cleaved. A fusion protein, HT*-FFP, and a Ca2+-dependent protease, CaTEV, will be co-expressed in neurons, both under constitutive promoters. Elevated Ca2+ will drive proteolytic cleavage of HT*, leading to incorporation of dark bands in the filament (HT* labels within the filament will be protected from proteolysis by the surrounding crystal). The outcome of this work will be an in vitro proof of concept of a new approach to brain-wide neural recording which does not require electrodes or optical access to the live tissue.

项目摘要/摘要 蛋白质的蛋白质tapes，用于整个大脑的神经记录 行为来自广泛分布的神经元集合的相互作用活动。但是所有现有工具 为了测量大脑活动样品，仅这些动力学的一小部分。在这里，我们提出了基于蛋白质的 记录两种神经活动的两个关键措施的大脑动力学方法：立即的早期基因（IEG） 表达和Ca2+浓度。该提案重点介绍了体外概念证明。后续工作将集中 如果有保证，则在体内应用程序上。 树戒指和股票磁带。今天在树上采取的核心可以揭示过去几个世纪以来的气候事件。 本提案适应了这一想法以记录神经激活的历史。这个想法是慢慢形成 生长的细胞内蛋白质丝的位置依赖性颜色反映了IEG活性或Ca2+的历史 动力学。高分辨率成像外体读取了这一历史。实现此目标的步骤是： 1）工程蛋白纤维作为分子式胶带。 Halotag受体（HT）将融合到 形成细胞中线性生长的结构的细丝形成蛋白（FFP）。连续添加不同的 彩色的脑部佩戴式吊带染料将在生长的细丝中产生彩色条纹，并映射纤维生长 到墙上的时间。蛋白质工程将用于优化成核，生长，标签和机械 这些纤维的特性。 2）记录IEG活性在蛋白质纤维上。 HT-FFP基因将在本构中表达 IEG启动子下的启动子和EGFP-FFP。 IEG激活将导致纤维中的绿色条纹，其绿色条纹 定时将通过参考不同颜色的Halotag条纹确定。启动子和蛋白质稳定性 将进行优化以实现高分辨率和长时间的IEG录音。 3）记录蛋白质纤维上的Ca2+动力学。 HT受体将被设计为包含TEV 蛋白酶识别基序（称为HT*），在切割时会使HT失活。融合蛋白，ht*-ffp和 Ca2+依赖性蛋白酶CATEV将在构成启动子下共表达神经元。 升高的Ca2+将驱动HT*的蛋白水解裂解，从而导致黑暗带掺入细丝中（HT* 细丝内的标签将受到周围晶体的蛋白水解保护。 这项工作的结果将是一种在整个大脑神经方法的概念的体外证明 记录不需要电极或光学进入活组织的记录。

项目成果

Time-tagged ticker tapes for intracellular recordings.

• DOI: 10.1038/s41587-022-01524-7
• 发表时间: 2023-05
• 期刊: NATURE BIOTECHNOLOGY
• 影响因子: 46.9
• 作者: Lin, Dingchang;Li, Xiuyuan;Moult, Eric;Park, Pojeong;Tang, Benjamin;Shen, Hao;Grimm, Jonathan B. B.;Falco, Natalie;Jia, Bill Z. Z.;Baker, David;Lavis, Luke D. D.;Cohen, Adam E. E.
• 通讯作者: Cohen, Adam E. E.