Development of kinase biosensors for multiplex neuronal imaging of signaling pathways in behaving mice

开发用于行为小鼠信号通路多重神经元成像的激酶生物传感器

• 批准号: 10505852
• 负责人: Richard L Huganir
• 金额: $ 240.98万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10505852
• 关键词: Address; Animals; BRAIN initiative; Benchmarking; Biosensor; Brain; Calcium; Cells; Color; Complex; Cyclic AMP-Dependent Protein Kinases; Development; Directed Molecular Evolution; Disease; Electroporation; Engineering; Event; Fluorescence Resonance Energy Transfer; Fluorescence-Activated Cell Sorting; Glutamate Receptor; Goals; Health; Image; Imaging Techniques; In Vitro; Intellectual functioning disability; Investigation; Laboratories; Learning; Locomotion; Long-Term Depression; Long-Term Potentiation; Mediating; Memory; Mental disorders; Molecular; Monitor; Mus; Neuromodulator; Neuronal Plasticity; Neurons; Noise; Pathway interactions; Performance; Pharmacology; Phosphotransferases; Photons; Physiological; Play; Process; Property; Protein Kinase; Proteins; Regulation; Research Proposals; Rewards; Role; Schizophrenia; Sensory; Signal Pathway; Signal Transduction; Slice; Synapses; Synaptic plasticity; System; Transfection; Validation; Variant; Visual Cortex; autism spectrum disorder; awake; base; calmodulin-dependent protein kinase II; design; experience; experimental study; fluorophore; in utero; in vivo; in vivo imaging; motor learning; nervous system disorder; neuronal excitability; next generation sequencing; novel; postsynaptic; sensor; tool; two photon microscopy; two-photon; voltage

Project Summary Cell signaling pathways in the brain are an essential part of a complex system regulating the activity and coordination of neuronal networks. During learning and memory these neuronal networks can be modified through neuronal and synaptic plasticity processes in which information is stored in the synaptic network. Intracellular signaling pathways play critical roles in regulating neuronal excitability and synaptic strength, thereby comprising an important part of the cellular and molecular mechanisms underlying learning and memory. Disruptions in the proper regulation of synaptic plasticity are involved in a number of neurological and psychiatric disorders including autism, schizophrenia, and intellectual disability. Revealing the dynamic activities and interactions of different signaling pathways is therefore crucial for understanding the mechanisms controlling neuronal networks both in health and disease. However, direct interrogation of signaling pathway activity in live animals has been challenging due to a lack of appropriate tools. Monitoring of multiple signaling pathways in such a setting has not been achieved. The goal of this research proposal is to develop novel tools to simultaneously monitor the activity of several signaling pathways and to use rapid, sensitive in vivo imaging techniques to visualize dynamic activity of these signaling pathways in live animals during physiologically relevant sensory experience and learning. Most existing biosensors for signaling activities are based on fluorescence resonance energy transfer (FRET) and the use of two different fluorescent proteins, which limits their use for monitoring of multiple signaling pathways in parallel. In this proposal, new single-color fluorescent protein-based kinase biosensors with high sensitivity and optimized two-photon excitation properties will be developed for imaging signaling pathways involved in synaptic plasticity, especially PKA, CaMKII, ERK, and PKC. The ultimate goal of this research proposal is to establish the use of these new biosensors in the mouse brain and to monitor both rapid dynamics of signaling pathways on the order of seconds to minutes and the long- term stability of signaling pathways on the order of weeks to months using two-photon microscopy in awake behaving animals. This proposed project will be the first investigation of multiple neuronal activities beyond calcium and voltage changes in live animals. These studies will allow us to examine the regulation of kinase pathways in vivo and will help elucidate the complexity of signaling pathways during synaptic plasticity in the brain.

项目摘要 大脑中的细胞信号传导通路是调节活动的复杂系统的重要组成部分， 神经网络的协调。在学习和记忆过程中，这些神经元网络可以被修改， 通过神经元和突触可塑性过程，其中信息存储在突触网络中。 细胞内信号通路在调节神经元兴奋性和突触强度中起着关键作用， 从而构成学习和记忆的细胞和分子机制的重要部分。 突触可塑性正常调节的中断涉及许多神经和精神疾病， 包括孤独症、精神分裂症和智力残疾在内的疾病。揭示动态活动， 因此，不同信号通路的相互作用对于理解控制 健康和疾病中的神经网络。然而，直接询问信号通路活动在生活中， 由于缺乏适当的工具，动物一直是一个挑战。多种信号通路的监测 这样的设置还没有实现。这项研究计划的目标是开发新的工具， 同时监测几种信号通路的活性，并使用快速，灵敏的体内成像 技术来可视化这些信号通路在生理过程中在活体动物中的动态活性， 相关的感官体验和学习。大多数现有的用于信号传导活动的生物传感器是基于 荧光共振能量转移（FRET）和使用两种不同的荧光蛋白，这限制了 它们用于并行监测多个信号传导途径的用途。在这个建议中，新的单色荧光 具有高灵敏度和优化的双光子激发特性的基于蛋白质的激酶生物传感器将 开发用于成像参与突触可塑性的信号通路，特别是PKA，CaMKII，ERK， 蛋白激酶C这项研究提案的最终目标是在小鼠中使用这些新的生物传感器 大脑，并监测信号通路的快速动态，时间为秒到分钟， 在清醒状态下使用双光子显微镜观察信号通路的长期稳定性， 行为动物这个拟议的项目将是第一次调查多种神经元活动超越 钙和电压的变化。这些研究将使我们能够检查激酶的调节 在体内的信号通路，并将有助于阐明在突触可塑性的信号通路的复杂性， 个脑袋