Spatiotemporal proteomic characterization of the human subventricular zone

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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.