Profiling activity-dependent synaptic translation

分析活动依赖性突触翻译

• 批准号: 9806853
• 负责人: NICHOLAS T INGOLIA
• 金额: $ 42.07万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9806853
• 关键词: Biotin; Biotinylation; Brain; Calcium; Cells; Codon Nucleotides; Coupled; Data; Dendrites; Disease; Disease model; Engineering; Expression Profiling; FMR1; FMRP; Fragile X Syndrome; Gene Expression; Goals; Hippocampus (Brain); Interneurons; Knockout Mice; Learning; Ligase; Light; Light Exercise; Link; Location; Long-Term Depression; Maps; Measures; Memory; Messenger RNA; Metabotropic Glutamate Receptors; Methods; Molecular; Mus; Neurobiology; Neurodevelopmental Disorder; Neurons; Neuropil; Pattern; Play; Process; Protein Biosynthesis; Proteins; Regulation; Regulator Genes; Resolution; Ribosomes; Role; Shapes; Signal Pathway; Signal Transduction; Synapses; Synaptic plasticity; System; Techniques; Transcript; Translating; Translational Regulation; Translations; Viral; Work; autism spectrum disorder; base; deep sequencing; experimental study; in vivo; innovation; insight; neural circuit; optogenetics; response; ribosome profiling; spatiotemporal; tool; translatome

ABSTRACT Synaptic plasticity allows for dynamic changes in the strength of neuronal connections, a hallmark of neural circuits that is thought to underlie learning and memory. Activity-dependent plasticity can strengthen or weaken synapses in response to calcium influx and other signaling pathways triggered by neuronal activity. These signals drive short- and long-term changes in synaptic strength through a variety of mechanisms. Local translation at active synapses is required for many long-term changes, likely through the synthesis of synaptic proteins. Subtle dysregulation of synaptic protein synthesis is thought to explain the neurodevelopmental features of fragile X syndrome, and is linked more broadly with autism spectrum disorders. These findings highlight the importance of regulated synaptic translation. Synaptic activity is transient, however, and active synapses are interspersed amongst many other synapses and cell bodies, posing serious technical challenges to profiling synaptic translation. Here, we propose to overcome these limitations and achieve global translational profiling at active synapses. We demonstrate a system for "tagging" ribosomes at active synapses using an engineered, calcium-dependent biotin ligase. Purification of these tagged ribosomes enriches for synaptic transcripts and confirms a key role for FMRP as a regulator of synaptic translation. In order to gain tighter spatiotemporal control needed for in vivo experiments, we have further engineered an optogenetic light-dependent biotin ligase substrate. Based on these promising preliminary data, we propose to develop and apply our calcium and light-dependent ribosome biotinylation to investigate activity-dependent translation. Results from this work will provide new insights into synaptic translation and produce widely applicable tools for neurobiology.

摘要 突触可塑性允许神经元连接强度的动态变化， 神经回路的一个标志，被认为是学习和记忆的基础。活性依赖 可塑性可以加强或削弱突触对钙离子和其他信号的反应 由神经元活动触发的通路。这些信号推动了短期和长期的变化， 突触强度通过各种机制。活跃突触的局部翻译是 这是许多长期变化所必需的，可能是通过合成突触蛋白。微妙 突触蛋白质合成的失调被认为可以解释神经发育障碍。 脆性X综合征的特征，并与自闭症谱系障碍有更广泛的联系。 这些发现强调了调节突触翻译的重要性。突触活动是 然而，短暂的和活跃的突触散布在许多其他突触之间， 细胞体，对突触翻译的研究提出了严峻的技术挑战。 在这里，我们建议克服这些限制，实现全球翻译轮廓， 活跃的突触我们展示了一个系统，用于“标记”核糖体在活跃的突触使用一个 工程化的钙依赖性生物素连接酶。这些标记的核糖体的纯化富集了 的突触转录本和确认FMRP作为突触翻译的调节器的关键作用。 为了获得体内实验所需的更严格的时空控制，我们进一步 工程化光遗传学光依赖性生物素连接酶底物。 基于这些有希望的初步数据，我们建议开发和应用我们的钙和 光依赖性核糖体生物素化以研究活性依赖性翻译。结果 这项工作将为突触翻译提供新的见解，并产生广泛适用的工具。 神经生物学。