Proteomic Screening of Neuronal and Glial Subcellular Compartments Isolated by Proximity Labeling and FACS in Fixed and Fresh Brain Samples

Neuronal subcellular compartments, such as dendritic spines, growth cones and synaptosomes, are critical for neuronal function in health and diseases. Glial subcellular constituents, such as myelin and astrocytic endfeet, support neuronal functions and maintain brain homeostasis. However, the molecular mechanisms and signaling pathways present in these subcellular compartments are not fully understood. Limited neuronal and glial subcellular proteomics data have been reported and the current protocols are mainly restricted to fresh samples, which pose barriers to study postmortem human and mouse pathologies with fixed tissue.

In this grant, we aim to implement and perfect methods for subcellular fraction isolation and proteomics methodologies with the goal of studying neuronal and glial subcellular compartments in both fresh and fixed mouse/human brain samples. Ultimately, our goal is to understand molecular mechanisms and signaling pathways governing subcellular compartments in diseases and health. Firstly, we innovatively apply a recently developed method called proximity labeling by using an in situ antibody recognition approach in fixed mice and human brain sections. With this approach, we can label and isolate endogenous subcellular proteins specifically and efficiently in a wide range of neuronal and glial subcellular compartments for subsequent label free LC-MS/MS proteomic analysis. Secondly, in order to compare proteome datasets generated by different isolation approaches, we will establish a Percoll gradient-FACS approach to isolate axonal subcellular compartments in fresh mouse brains, followed by label free LC-MS/MS. Comparison of proteome datasets from proximity labeling with those from FACS will provide cross validation of these methods of neuronal and glial subcellular protein analysis. Thirdly, we have established sophisticated optical imaging toolsets to validate proteomic hits in both fixed samples and in vivo that will be implemented to further study protein targets identified by proteomics in both mouse models and human postmortem tissue, in the context of the axonopathy found around amyloid plaques in Alzheimer’s disease (AD). This proposal is of high impact because it will perfect and implement methodologies that are widely useful in neuroscience research. In addition, the results obtained from the proteomics analysis could have important implications on our understanding of AD pathophysiology and treatment design.