Spatiotemporal proteomic characterization of the human subventricular zone

背景：揭示驱动人类大脑发育的分子程序仍然是神经科学的最后前沿之一。虽然跨物种保守的基本过程现在已经阐明，但在人类和高等灵长类动物中运作的神经生物学机制仍有待解决。这包括驱动人类脑室下区（SVZ）大规模扩张和持续神经元输出的分子程序。然而，这些过程发生在发展的早期，基本上无法进行纵向监测。在这里，我们的目标是通过整合液相色谱-串联质谱（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。我们的具体目标是：目标1：在人类神经发育过程中定义SVZ内的时空蛋白质组差异AIM 2：解密SVZ富集蛋白的机制和功能AIM 3：解决SVZ富集蛋白的亚结构水平共分布模式方法：我们利用临床样本的访问来组装一组福尔马林固定石蜡包埋（FFPE）胎儿脑标本，这些标本来自神经发育不同阶段的存档尸检材料。这包括28个大脑，跨越妊娠周（GW）15 - 29之间的4个不同时期（6 - 8个大脑/时间段）。对AIM 1，我们耦合激光捕获显微切割和无标记的定量LC-MS/MS来定义SVZ及其下游神经元隔室的主要细分内的时空蛋白质组模式。对于AIM2，我们利用脑类器官培养系统来解决潜在的表型效应和机制，这些表型效应和机制是我们在神经发生高峰期（GW 15 - 17）通过LC-MS/MS鉴定的有希望的SVZ富集候选物（CLIC 1，ANXA 5，PLIN 3，FLNC）。这包括当用化学/遗传探针操纵候选蛋白质时类器官中的表型细胞和分子变化。最后，我们开发了一个深度学习分析管道，以确定SVZ特异性蛋白质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早期人类神经发育过程中运作的分子程序。首先，我们将生成第一个空间保存的蛋白质水平的人类SVZ图谱。我们补充这一资源与功能的研究SVZ样地区的三维组织样脑类器官文化建模。我们的目标是通过使用计算机视觉工具时空解析有前途的SVZ富集蛋白质的共分布来进一步丰富这个数据集。这种时空分析一起用于开始表征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.