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

Abstract

Proteomic profiling of brain cell types using isolation-based strategies pose limitations in resolving cellular phenotypes representative of their native state. We describe a mouse line for cell type-specific expression of biotin ligase TurboID, for in vivo biotinylation of proteins. Using adenoviral and transgenic approaches to label neurons, we show robust protein biotinylation in neuronal soma and axons throughout the brain, allowing quantitation of over 2000 neuron-derived proteins spanning synaptic proteins, transporters, ion channels and disease-relevant druggable targets. Next, we contrast Camk2a-neuron and Aldh1l1-astrocyte proteomes and identify brain region-specific proteomic differences within both cell types, some of which might potentially underlie the selective vulnerability to neurological diseases. Leveraging the cellular specificity of proteomic labeling, we apply an antibody-based approach to uncover differences in neuron and astrocyte-derived signaling phospho-proteins and cytokines. This approach will facilitate the characterization of cell-type specific proteomes in a diverse number of tissues under both physiological and pathological states.

Distinguishing biophysical stochasticity from technical noise in single-cell RNA sequencing usingMonod

“…We omit the discussion of A230006K03Rik, Camk2a, Satb2, Sv2b , and Prkce , as their fits are quite poor. Camk2a demonstrates considerable multimodality, likely due to cell subtype localization or incorrect assignment of Camk2a neurons to the GABAergic class [45]. Sv2b may show multimodality due to sequencing or alignment ambiguities between its two major isoforms [46].…”

Cellular proteomic profiling using proximity labelling by TurboID-NES in microglial and neuronal cell lines

“…The viability of the latter application was recently tested in vivo using genetic Cre/lox strategies to resolve neuronal and astrocyte proteomes in the native state of these cells in mouse brain, a method referred to as cell type-specific in vivo biotinylation of proteins (CIBOP). 10 Whether these neuronal or glial proteomes obtaining using CIBOP reflect global cellular proteomes, remains to be clarified. As interest in TurboID-based global cellular proteomics continues to grow, it is important to determine what fraction of the whole cell proteome in mammalian cells can be faithfully captured by the TurboID-NES approach, under both homeostatic (resting) conditions and following cellular perturbations (eg.…”

“…Slightly modified from previous publications the AP samples were processed as follows: 13, 22 1 mg of protein derived from transduced and untransduced BV2 and N2A lysates were affinity-purified onto 83μL of magnetic streptavidin beads (Thermo #88817). Briefly, to each 1.5 mL Eppendorf LoBind tube, 1 mL of RIPA buffer (150 mM NaCl, 50mM Tris, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, pH 7.5) was added to the beads on rotation for 2 minutes at room temperature (RT).…”

“…Immunofluorescent staining was performed as published previously with modifications for cultured cells. 10 Briefly, BV2 and N2A cells were seeded at ~50,000 cells onto autoclaved and HCl-ethanol-treated 25mm coverslips in a 6-well dish. All cells received supplemental biotin treatment (200μM) for the duration of maintenance.…”

“…Slightly modified from previous publications the AP samples were processed as follows: 13,22 1 mg of protein derived from transduced and untransduced BV2 and N2A lysates were affinity-purified onto 83μL of magnetic streptavidin beads (Thermo #88817).…”