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.
Highlights
The vertebrate brain consists of diverse neuronal types, classified by distinct anatomy and function, along with divergent transcriptomes and proteomes
We demonstrated Cre-dependent expression by neonatal viral transduction in the striatum of Drd1Cre mice with adeno-associated viral vectors (AAVs) encoding one of three APEX variants and a EGFP reporter with a ribosomal skipping P2A linker (Fig. 1a)
In all APEX variants (Fig. 1b and Supplementary Fig. 1a) EGFP expression was similar across constructs, as expected for P2A-linked EGFP, where its distribution is independent from the APEX localization and should be the same regardless of APEX targeting
Summary
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. In many laboratory model systems, identifying distinct cell classes in the brain is achieved by conditional expression of sitespecific recombinases and fluorescent reporters, or other effectors, under the control of a gene-specific promoter[3] Cell isolation approaches such as laser capture microdissection (LCM)[4] or tissue dissociation followed by fluorescent cell sorting[5] are usually employed to isolate reporter-positive somata for proteomics analyses. Because the incorporation of NCAAs depends on protein translation, this approach is not suitable for answering questions about many rapid biological processes: the experimental time window of NCAA incorporation can take up to several days[8,9]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
