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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要用于全脑神经记录的蛋白质自动收报机磁带行为是由广泛分布的神经元群的相互作用活动产生的。但所有现有的工具为了测量大脑活动，仅采样这些动态的一小部分。在这里，我们提出了一种基于蛋白质的记录神经活动两个关键指标的全脑动态的方法：即早基因（IEG）表达和 Ca2+ 浓度。该提案重点关注体外概念验证；后续工作将重点如果有必要的话，在体内应用。树木年轮和自动收报机磁带。今天从树上采集的核心可以揭示过去几个世纪的气候事件。目前的提案采用了这个想法来记录神经激活的历史。想法是慢慢形成的生长的细胞内蛋白丝，其位置依赖性颜色反映了 IEG 活性或 Ca2+ 的历史动力学。高分辨率离体成像读取了这段历史。实现这一目标的步骤是： 1) 将蛋白质纤维设计为分子收报机磁带。 HaloTag 受体 (HT) 将融合到丝状形成蛋白（FFP）在细胞中形成线性生长的结构。依次添加不同的彩色脑渗透性 HaloTag 染料将在生长的细丝中形成彩色条纹，绘制纤维生长图到挂钟时间。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基因将在组成型下表达启动子和 IEG 启动子下的 eGFP-FFP。 IEG 激活将导致纤维中出现绿色条纹，其计时将根据不同颜色的 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Ca2+动态。 HT 受体将被设计为包含 TEV 蛋白酶识别基序（称为 HT*），在裂解时使 HT 失活。融合蛋白 HT*-FFP，和 Ca2+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的作用下。升高的 Ca2+ 将驱动 HT* 的蛋白水解裂解，导致细丝中暗带的掺入 (HT* 细丝内的标签将受到周围晶体的保护，免受蛋白水解）。这项工作的成果将是一种新的全脑神经方法的体外概念证明。记录不需要电极或光学进入活组织。

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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.