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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 generated a novel mouse line for cell type-specific expression of biotin ligase TurboID, leading to in vivo biotinylation of proteins. Using adenoviral and transgenic approaches to label neurons, we show striking protein biotinylation in neuronal soma and axons throughout the brain and quantified over 2,000 neuron-derived proteins spanning synaptic proteins, transporters, ion channels and disease-relevant druggable targets. Next, we compared Camk2a-neuron and Aldh1l1-astrocyte proteomes and resolved brain region-specific proteomic differences within both cell types, some of which may underlie selective vulnerability to neurological diseases. Leveraging the cellular specificity of proteomic labeling, we used 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Duc Duong

LAB HEAD: Srikant Rangaraju

PROVIDER: PXD032161 | Pride | 2022-04-28

REPOSITORIES: Pride

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			Action	DRS
	checksum.txt	Txt
	ip_maxquant_txtfolder.zip	Other
	region_maxquant_txtfolder.zip	Other
	sruti_56ips_11052021_ip01.raw	Raw
	sruti_56ips_11052021_ip02.raw	Raw

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Project description:Aims: This study aimed to identify and characterize the intrinsic properties of locus coeruleus (LC) noradrenergic neurons in male and female mice using a genetic approach. We also sought to investigate sex-specific differences in membrane properties, action potential generation, and protein expression profiles to understand the mechanisms underlying neuronal excitability variations. Methods: Utilizing a genetic mouse model by crossing Dbhcre knock-in mice with tdTomato Ai14 transgenic mice, LC neurons were identified using fluorescence microscopy. Neuronal properties, including capacitance, action potential frequency, and rheobase, were assessed using patch-clamp recordings. Spontaneous and evoked activity between sexes was compared. Proteomic analyses of individual LC neuron soma was conducted using mass-spectrometry to discern protein expression profiles. Results: Female LC noradrenergic neurons displayed greater membrane capacitance than those in male mice. Male LC neurons demonstrated greater spontaneous and evoked action potential generation compared to females. Male LC neurons exhibited a lower rheobase and achieved higher peak frequencies with similar current injections. Proteomic analysis revealed inherent differences in protein expression profiles between sexes, with male mice displaying a notably larger unique protein set compared to females. Notably, pathways pertinent to protein synthesis, degradation, and recycling, such as EIF2 and glucocorticoid receptor signaling, showed reduced expression in females. Conclusions: Male LC noradrenergic neurons exhibit higher intrinsic excitability compared to those from females. The discernible sex-based differences in excitability could be ascribed to varying protein expression profiles, especially within pathways that regulate protein synthesis and degradation. This study lays the groundwork for future studies focusing on the interplay between proteomics and neuronal function examined in individual cells.

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

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