Spatiotemporal proteomic characterization of the human subventricular zone

背景：解开驱动人脑发育的分子程序仍然是神经科学的最后前沿之一。虽然现在已经阐明了跨物种保守的基本过程，但在人类和高等灵长类动物中运行的神经生物学机制仍有待解决。这包括推动人类脑室下区(SVZ)大规模扩张和持续神经元输出的分子程序。然而，这些进程发生在发展的早期，很大程度上无法进行纵向监测。在这里，我们旨在通过整合LC-MS/MS(LC-MS/MS)、组织生物工程和深度学习的专业知识来解决这一差距，以绘制发育中的人类SVZ的时空蛋白质组结构图。我们的具体目标是：AIM1：确定人类神经发育过程中SVZ区内的时空蛋白质组差异AIM2：破译SVZ富集区蛋白质的机制和功能AIM3：解析SVZ富集区蛋白质亚结构水平的共分布模式方法：我们利用对临床样本的获取来收集来自神经发育不同阶段的档案尸检材料的福尔马林固定石蜡包埋(FFPE)胎儿大脑样本。这包括28个大脑，跨越4个不同的孕周(GW)15-29(6-8个大脑/时间段)。对于AIM1，我们结合激光捕获显微切割和无标记LC-MS/MS来定义SVZ主要亚区及其下游神经元室内的时空蛋白质组模式。对于AIM2，我们利用脑器官培养系统来解析我们在神经发生高峰期(GW15-17)通过LC-MS/MS鉴定的有希望的SVZ富集型候选基因(CLIC1、ANXA5、PLIN3、FLNC)的潜在表型效应和机制。这包括当候选蛋白质被化学/遗传探针操纵时，有机物中的表型、细胞和分子的变化。最后，我们开发了一个深度学习分析管道来精确定位SVZ特定蛋白的亚结构水平免疫组织化学共分布染色模式。影响：对非灵长类动物大脑的广泛研究突出了神经发育的基本过程。在这里，我们利用一个独特的人类组织队列，并应用新的和新兴的技术来定义在人类早期神经发育期间可操作的分子程序。首先，我们将生成第一个空间保存的人类SVZ蛋白质水平图谱。我们通过在三维组织样脑器官培养中模拟的SVZ样区的功能研究来补充这一资源。我们的目标是通过使用计算机视觉工具在时空上解析有前景的SVZ富集蛋白的共同分布来进一步丰富这个数据集。总而言之，这种时空分析开始描述人脑下游分子租户的结构和功能。

BACKGROUND: Unraveling the molecular programs driving human brain development remains one of the final frontiers in neural sciences. While fundamental processes conserved across species have now been elucidated, neurobiological mechanisms operational in humans and higher primates still remain to be resolved. This includes the molecular programs driving the massive expansion and sustained neuronal output of the human subventricular zone (SVZ). These processes however, occurring early in development, have been largely inaccessible for longitudinal monitoring. Here, we aim to address this gap by integrating expertise in liquid chromatography-tandem mass spectrometry (LC-MS/MS), tissue bioengineering and deep learning to map the spatiotemporal proteomic architecture of the developing human SVZ. We specifically aim to: AIM1: Define spatiotemporal proteomic differences within the SVZ during human neurodevelopment AIM2: Decipher mechanisms and function of SVZ-enriched proteins AIM3: Resolve substructure-level co-distribution patterns of SVZ-enriched proteins APPROACH: We leverage access to clinical samples to assemble a cohort of formalin-fixed paraffin-embedded (FFPE) fetal brain specimens from archival autopsy material at different stages of neurodevelopment. This includes 28 brains spanning 4 distinct periods between gestational weeks (GW) 15-29 (6-8 brains/time period). Towards AIM1, we couple laser capture microdissection and label-free quantitative LC-MS/MS to define spatiotemporal proteomic patterns within the major sub-divisions of the SVZ and their downstream neuronal compartments. Towards AIM2, we take advantage of the cerebral organoid culture system to resolve potential phenotypic effects and mechanisms of promising SVZ-enriched candidates (CLIC1, ANXA5, PLIN3, FLNC) we identified by LC-MS/MS during the peak of neurogenesis (GW15-17). This include phenotypic cellular and molecular changes in organoids when candidate proteins are manipulated with chemical/genetic probes. Finally, we develop a deep learning analytical pipeline to pinpoint sub-structure-level immunohistochemical co-distribution staining patterns of SVZ-specific proteins. IMPACT: Extensive study of the non-primate brain has highlighted fundamental processes of neurodevelopment. Here, we take advantage of a unique human tissue cohort and apply new and emerging technologies to define molecular programs operational during early human neurodevelopment. Firstly, we will generate the first spatially-preserved protein-level atlas of the human SVZ. We complement this resource with functional studies in SVZ-like regions modeled in tridementional tissue-like cerebral organoid cultures. We aim to further enrich this dataset by spatiotemporally resolving the co-distribution of promising SVZ-enriched protein using computer vision tools. Together this spatiotemporal analysis serves to begin characterizing the architecture and function of the downstream molecular tenants of the human brain.