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.

项目总结/摘要 全脑神经记录的蛋白质自动收报机 行为来自广泛分布的神经元集合的相互作用活动;但所有现有的工具 为了测量大脑活动，仅对这些动态的一小部分进行采样。在这里，我们提出了一种基于蛋白质的 一种记录神经活动的两个关键指标的全脑动态的方法：立即早期基因（IEG） 表达和Ca 2+浓度。该提案侧重于体外概念验证;后续工作将侧重于 在体内应用，如果需要的话。 树木年轮和股票行情。今天在一棵树上采集的核心可以揭示几个世纪前的气候事件。 目前的建议采用这种想法来记录神经激活的历史。我们的想法是慢慢形成 生长的细胞内蛋白丝，其位置依赖性颜色反映IEG活性或Ca 2+的历史 动力学离体高分辨率成像读取这段历史。实现这一目标的步骤是： 1)工程蛋白质纤维作为分子自动收报机。HaloTag受体（HT）将与 在细胞中形成线性生长结构的蛋白质（FFP）。顺序添加不同的- 着色的脑渗透性HaloTag染料将在生长的细丝中产生彩色条纹，映射纤维生长 到挂钟时间蛋白质工程将用于优化成核，生长，标记和机械 这些纤维的特性。 2)记录蛋白质纤维上的IEG活性。HT-FFP基因将在组成型表达下表达。 启动子和IEG启动子下的eGFP-FFP。IEG激活将导致光纤中出现绿色条纹， 定时将通过参考不同颜色的HaloTag条纹来确定。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基IEG记录。 3)记录蛋白质纤维上的Ca 2+动力学。HT受体将被改造成含有TEV 蛋白酶识别基序（称为HT*），其在切割时使HT失活。融合蛋白HT*-FFP，和 Ca 2+依赖性蛋白酶CaTEV将在神经元中共表达，两者都在组成型启动子下。 升高的Ca 2+将驱动HT* 的蛋白水解裂解，导致暗带掺入细丝（HT* 细丝内的标记将受到周围晶体的保护而不被蛋白水解）。 这项工作的结果将是一种新的脑神经细胞移植方法的体外概念证明。 不需要电极或光学通路进入活组织的记录。

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.