Proteome-Driven Holistic Reconstruction of Organ-Wide Multi-Scale Networks

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Abstract Human organs such as the brain are stunningly complex. They consist of hundreds to thousands of separate functional areas, each containing a comparable number of distinct cell types and innumerable molecules. Understanding how these multi-scale components work together to generate systems-level responses is essential for many fields of biology, but advancement in this area is hampered by the prevalent methodology of dividing biological systems into known cell types and then separately studying each population. Although powerful, this reductionistic approach makes it difficult to interrogate complex interactions at multiple levels — molecular (e.g., proteins), subcellular (e.g., synapses), cellular, and area level. Moreover, this approach could ignore many potentially important but unidentified functional networks. Our inability to thoroughly identify multi- scale functional networks and interrogate their system-wide, multifactorial interactions has limited our ability to understand the function and dysfunction of complex biological systems. Here, we aim to fundamentally transform our approach from a reductionistic to a holistic one by developing cutting-edge platforms for proteomic reconstruction of organ-wide multi-scale networks. Using murine and human clinical samples and organoids as our models, we will develop four broadly applicable cross-disciplinary platforms that integrate chemical and material engineering technologies. These platforms will enable: (1) scalable tissue transformation into an indestructible, proteome-containing three-dimensional (3D) framework; (2) unlimited rounds of molecular phenotyping of a single intact tissue with precise volume co-registration of multiple datasets; (3) rapid, scalable, and uniform tissue labeling by synchronizing target-probe binding reactions organ-wide; (4) superresolution proteomic imaging of intact organs. If successful, our proposed work will enable proteome- driven holistic reconstruction and high-dimensional quantitative phenotyping of intact biological systems at unprecedented resolution. Using the technology platforms and human brain organoid models, both healthy and diseased, we will investigate the following fundamental questions: (Q1) How many cell types/regions exist at different developmental stages of the brain organoids? (Q2) How these cells and regions form networks? (Q3) How proteomic states of subcellular components, individual cells, circuits, and regions change throughout the development. (Q4) How morphological features of cells change? (Q5) how Q1-4 are altered in diseased organoids? This study may provide new insights into understanding human organ development in health and disease.