Activity-dependent ribosome profiling reveals the landscape of canonical and non-canonical translation in brain tissue [Ribo-Seq]

Neural activity-dependent translation is essential for synaptic plasticity and diverse brain functions. Translation involves not only canonical main open reading frames (mORFs) but also upstream ORFs (uORFs), which may regulate mORF expression. However, due to technical limitations, systematic in-vestigation of activity-dependent uORFs and mORFs in brain tissues remains challenging. Here, we developed a ribosome tagging and purification strategy that bypasses the prolonged turnover of ribo-somal proteins, enabling ribosome profiling with one-hour temporal resolution after neural stimulation. Applying this strategy to mouse hippocampal slices undergoing long-term potentiation, we identified hundreds of activity-induced mORFs and uORFs, a subset of which served as robust markers for ac-tivity status. Notably, ~22% of the upregulated uORFs overlapped with those induced by the integrated stress response, suggesting potential crosstalk between these signaling pathways. This study provides a useful technique and resources for deciphering molecular mechanisms underlying activity- and trans-lation-dependent brain functions in health and disease. Overall design: Ribo-seq, Postnatal day 5 mice Organotypic hippocampus slice culture

依赖神经活动的翻译过程对于突触可塑性及多种脑功能至关重要。翻译过程不仅涉及经典主要开放阅读框（canonical main open reading frames, mORFs），还包含上游开放阅读框（upstream ORFs, uORFs），后者可调控mORF的表达。然而，受限于现有技术手段，在脑组织中系统性研究依赖神经活动的uORFs与mORFs仍颇具挑战。本研究开发了一种可绕过核糖体蛋白长周期周转的核糖体标记纯化策略，能够在神经刺激后以1小时的时间分辨率开展核糖体谱分析。我们将该策略应用于处于长时程增强（long-term potentiation, LTP）状态的小鼠海马脑片，成功鉴定出数百种神经活动诱导的mORFs与uORFs，其中部分可作为神经活动状态的可靠标志物。值得注意的是，约22%的上调uORFs与整合应激反应（integrated stress response, ISR）诱导的uORFs存在重叠，提示这两条信号通路间可能存在交互调控。本研究为解析健康与疾病状态下依赖神经活动与翻译过程的脑功能分子机制，提供了实用的技术方法与研究资源。实验设计概述：Ribo-seq、出生后第5天小鼠、器官型海马脑片培养