Poster - #UCLA-26 - Keywords: astrocytes neurons plasma membrane proteomics

Identifying astrocyte and neuron plasma membrane proteins in vivo

Joselyn S. Soto, Yasaman Jami-Alahmadi, James A. Wohlschlegel, and Baljit S. Khakh
Department of Physiology, Department of Neurobiology, Department of Biological Chemistry David Geffen School of Medicine, UCLA

ABSTRACT:
Astrocytes and neurons both contribute to diseases of the nervous system. Astrocytes are bushy cells that tile the entire central nervous system (CNS) and contribute, along with neurons, to the formation of complex networks with dense physical and functional connectivity between these cell types. Traditional methods to investigate plasma membrane proteomes have used either primary cell cultures or cells that have been acutely separated from tissue and have been purified based on cell surface markers for analysis. However, the resulting cells are no longer physiological and lose their cellular integrity, associated proteins, as well as most of their native connectivity. Consequently, the in vivo proteome of astrocytes and neurons in normal or dysfunctional states remains largely unknown. Therefore, to understand the role of astrocytes and their interactions with neurons at the protein level, we have designed a cell-specific method using the biotin ligase, BioID2, to tag proteomes in astrocyte and neuron plasma membrane compartments in vivo. In short, this BioID2 system uses proximity-dependent biotin labeling to tag proximal proteins, which can then be purified and identified upon the addition of streptavidin, a strong biotin binder. In vivo validation shows that BioID2 expression within astrocytes and neurons can be restricted to the plasma membrane and demonstrates a high level of biotinylation upon the addition of biotin. Subsequent biotinylated protein isolation and identification with liquid chromatography-tandem mass spectrometry (LC-MS/MS) reliably shows major known proteins present in striatal astrocytes and neurons. Our method is the first to capture plasma membrane proteomes from genetically defined astrocytes and neurons while preserving native morphology and connectivity for downstream proteomic analyses. The use of this method will lead to the identification of new therapeutic targets in astrocytes and neurons that can be explored in multiple CNS disease states.