Cell type-specific biotin labeling in vivo resolves regional neuronal proteomic differences in mouse brain

Abstract

Isolation and proteomic profiling of brain cell types, particularly neurons, pose several technical challenges which limit our ability to resolve distinct cellular phenotypes in neurological diseases. Therefore, we generated a novel mouse line that enables cell type-specific expression of a biotin ligase, TurboID, via Cre-lox strategy for in vivo proximity-dependent biotinylation of proteins. Using adenoviral-based and transgenic approaches, we show striking protein biotinylation in neuronal cell bodies and axons throughout the mouse brain. We quantified more than 2,000 neuron-derived proteins following enrichment that mapped to numerous subcellular compartments. Synaptic, transmembrane transporters, ion channel subunits, and disease-relevant druggable targets were among the most significantly enriched proteins. Remarkably, we resolved brain region-specific proteomic profiles of Camk2a neurons with distinct functional molecular signatures and disease associations that may underlie regional neuronal vulnerability. Leveraging the neuronal specificity of this in vivo biotinylation strategy, we used an antibody-based approach to uncover regionally unique patterns of neuron-derived signaling phospho-proteins and cytokines, particularly in the cortex and cerebellum. Our work provides a proteomic framework to investigate cell type-specific mechanisms driving physiological and pathological states of the brain as well as complex tissues beyond the brain.

Dynamic proteomic and phosphoproteomic atlas of corticostriatal axons in neurodevelopment

“…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 ; Sun et al, 2022 ). 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 .…”

“…In comparison with biotin ligase-based reporters (e.g. BioID, TurboID), APEX-mediated labeling occurs within 1 hr ex vivo in acute brain sections, while in vivo BioID/TurboID labeling takes several days up to weeks, via continuous biotin supplementation ( Rayaprolu et al, 2021 ; Spence et al, 2019 ; Sun et al, 2022 ; Uezu et al, 2016 ). Therefore, the APEX-based approach is more suitable for biological questions that require short temporal windows, while BioID/TurboID can measure in vivo steady-state proteome spanning the duration of biotin administration.…”

“…Axons from different circuits and cell types are intermingled, and isolation techniques that lack genetic targeting likely mask neuronal class specific features. Several genetically encoded proteomics strategies have been developed to profile cell-type-specific proteomes in the mouse brain, including metabolic labeling ( Alvarez-Castelao et al, 2017 ; Krogager et al, 2018 ) and proximity labeling strategies ( Dumrongprechachan et al, 2021 ; Hobson et al, 2022 ; Rayaprolu et al, 2021 ; Spence et al, 2019 ; Uezu et al, 2016 ). We and others have previously shown that APEX-based proximity labeling in the acute brain sections is highly efficient and robust, providing sufficient material with both cell-type and subcellular compartment specificity such that there is no need for tissue dissociation, biochemical fractionation, or subject pooling ( Dumrongprechachan et al, 2021 ; Hobson et al, 2022 ).…”

Dynamic proteomic and phosphoproteomic atlas of corticostriatal axon neurodevelopment

“…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 .…”

“…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 . In comparison with biotin ligase-based reporters ( e.g ., BioID, TurboID), APEX-mediated labeling occurs within 1 hr ex vivo in acute brain sections, while in vivo BioID/TurboID labeling takes several days up to weeks, via continuous biotin supplementation (Rayaprolu et al, 2021; Spence et al, 2019; Uezu et al, 2016). Therefore, the APEX-based approach is more suitable for biological questions that require short temporal windows, while BioID/TurboID can measure in vivo steady-state proteome spanning the duration of biotin administration.…”