Cell-type and subcellular compartment-specific APEX2 proximity labeling reveals activity-dependent nuclear proteome dynamics in the striatum

Abstract

The vertebrate brain consists of diverse neuronal types, classified by distinct anatomy and function, along with divergent transcriptomes and proteomes. Defining the cell-type specific neuroproteomes is important for understanding the development and functional organization of neural circuits. This task remains challenging in complex tissue, due to suboptimal protein isolation techniques that often result in loss of cell-type specific information and incomplete capture of subcellular compartments. Here, we develop a genetically targeted proximity labeling approach to identify cell-type specific subcellular proteomes in the mouse brain, confirmed by imaging, electron microscopy, and mass spectrometry. We virally express subcellular-localized APEX2 to map the proteome of direct and indirect pathway spiny projection neurons in the striatum. The workflow provides sufficient depth to uncover changes in the proteome of striatal neurons following chemogenetic activation of Gαq-coupled signaling cascades. This method enables flexible, cell-type specific quantitative profiling of subcellular proteome snapshots in the mouse brain.

Subcellular proteomics of dopamine neurons in the mouse brain

“…Our study demonstrates APEX2 labeling and MS-based proteomics of axonal and somatodendritic compartments of mDA neurons in the mouse brain. Thus, APEX2 labeling in acute brain slices provides a general approach for cell type-specific proteomics and/or proximity labeling proteomics in the mouse brain (see also Dumrongprechachan et al, 2021 ), alongside promiscuous biotin ligase (BioID or TurboID)-catalyzed proximity labeling ( Takano et al, 2020 ; Uezu et al, 2016 ) and incorporation of non-canonical amino acids via mutant tRNA synthetases ( Alvarez-Castelao et al, 2017 ; Krogager et al, 2018 ). Each of these methods has advantages and disadvantages.…”

Dynamic proteomic and phosphoproteomic atlas of corticostriatal axon neurodevelopment

“…The unbound flowthrough was fractionated using the high-pH reverse fractionation to measure protein abundance. We adapted our previous analysis workflow using MSstatsTMT for protein summarization, and comparison with Cre-negative samples for removing non-specific binding contaminants (Dumrongprechachan et al, 2021). MSstatsTMT was used to summarize peptide intensity into protein abundance (Huang et al, 2020).…”

“…Genetically encoded proximity labeling is an effective way to perform cell-type and subcellular compartment specific proteomics in the mouse brain (Dumrongprechachan et al, 2021; Hobson et al, 2022; Rayaprolu et al, 2021). Here, we generated a new Cre-dependent APEX2 reporter line to capture snapshots of the neuroproteome with cell type specificity, illustrated by several genetic crosses including VGlut2 Cre , VGAT Cre , and Rbp4 Cre .…”

“…TMT labeled peptides (~1 μg) were resuspended in 2% acetonitrile/0.1% formic acid prior to being loaded onto a heated PepMap RSLC C18 2 μm, 100 angstrom, 75 μm x 50 cm column (ThermoScientific) and eluted over 180 min gradients optimized for each high pH reverse-phase fraction (Dumrongprechachan et al, 2021). Sample eluate was electrosprayed (2,000V) into a Thermo Scientific Orbitrap Eclipse mass spectrometer for analysis.…”

“…This protocol was modified from our previous publication (Dumrongprechachan et al, 2021). Total protein was extracted by sonication in lysis buffer (1% SDS, 125 mM TEAB, 75 mM NaCl, and Halt TM protease and phosphatase inhibitors).…”