CAREER: Mapping the proteomic landscape of neural systems.

Overview: The broad scientific objective of this proposal is to reveal the dynamic proteomic landscape of striatal neurons, relying on new tools we have developed for genetically targeted, quantitative bottom-up proteomics. There are no existing techniques for genetically targeted neuroproteomics in situ, within the native neural circuit or cellular context. The goal of this proposal is to introduce and use a powerful suite of techniques for unlocking the proteome of any cell type in the brain or in the body. Our approach leverages the power of peroxidases, enzymes that can be induced to biotinylate neighboring proteins. We will validate and use our new tool kit of flexible recombinant adeno-associated viral vectors (AAVs) that conditionally express Apex peroxidase throughout the cytosol or targeted to neuronal membranes. We will use our new tools to address critical questions in neurobiology about development and neuromodulation of cell types in the striatum. In addition, we will generate new, even more powerful proteomics tools by building a conditional Apex reporter mouse line for a lasting, transformative impact on biology and neuroscience. Objective 1. Map the proteomics landscapes of striatal spiny projection neurons (SPNs) across development. We hypothesize that the proteomic landscape of SPNs changes during development. SPNs dramatically alter neuronal excitability and structure over time, but proteomic correlates of neuronal maturation are unknown. Apex will be transduced in the two types of SPNs selectively, for label-free or tandem mass tag-based quantitative proteomics. Objective 2. Quantify the proteomic signature of SPN response to optogenetically evoked dopamine release. We hypothesize, based on preliminary data, that stimulating dopamine receptors on the two types of SPNs leads to large-scale changes in the abundance of proteins involved in synaptic function, axonal transport and neuronal plasticity. We will test this prediction by using conditional Apex viruses we have built and validated. Objective 3. Generate a Cre recombinase-dependent cytosolic proteomic reporter line. We will use CRISPR/Cas or traditional mutagenesis to generate a mouse carrying permissive locus knock-in of conditional Apex and GFP. This proteomic reporter will be made available for deep proteomic sampling of any Cre-expressing cell type (>1500 mouse lines) in the brain or in the body.