Landgraf, P. | Search bioRxiv

bioRxiv2022

Monitoring regional astrocyte diversity by cell-type specific proteomic labeling in vivo

Astrocytes exhibit regional heterogeneity in morphology, function and molecular composition to support and modulate neuronal function and signaling in a region-specific manner. To characterize regional heterogeneity of astrocytic proteomes of different brain regions we established an Aldh1l1- MetRSL274G mouse line that allows cell-type specific labeling of newly synthesized proteins in vivo and analyzed astrocytic proteins from four different brain regions by mass spectrometry. Identified proteins are specific for astrocytes and show a high overlap with proteins compiled in AstroProt, a newly established database for astrocytic proteins. Gene enrichment analysis reveals high overlap among brain regions, and only subtle changes in abundances of key astrocytic proteins for hippocampus, cortex and striatum. However, the cerebellar proteome stands out with proteins being associated with calcium signaling or bipolar disorder. Subregional differences of translation dynamics in single hippocampal astrocytes indicates distinct subregional heterogeneity and highlights the applicability of our toolbox to study dynamic astrocytic proteomes in vivo.

neuroscience↗

bioRxiv2020

SUMOylation regulates protein cargo in Astrocyte-derived small extracellular vesicles

Recent studies have described a new mechanism of intercellular communication mediated by various types of extracellular vesicles (EVs). In particular, exosomes are small EVs (sEVs) released to the extracellular environment by the fusion of the endosomal pathway-related multivesicular bodies (containing intraluminal vesicles) with the plasma membrane. sEVs contain a molecular cargo consisting of lipids, proteins, and nucleic acids. However, the loading mechanisms for this complex molecular cargo have not yet been completely elucidated. In that line, the post translational modification SUMO (Small Ubiquitin-like Modifier) has been shown to impact the incorporation of select proteins into sEVs. We therefore decided to investigate whether SUMOylation is a mechanism that defines protein loading to sEVs. In order to investigate the role of SUMOylation in cargo loading into sEVs, we utilized astrocytes, an essential cell type of the central nervous system with homeostatic functions, to study the impact of SUMOylation on the protein cargo of sEVs. Following SUMO overexpression, achieved by transfection of SUMO plasmids or experimental conditions that modulate SUMOylation in primary astrocyte cultures, we detected proteins related to cell division, translation, and transcription by mass-spectrometry. In astrocyte cultures treated with the general SUMOylation inhibitor 2-D08 (2',3',4'-trihydroxy-flavone, 2-(2,3,4-Trihydroxyphenyl)-4H-1-Benzopyran-4-one) we observed an increase in the number of sEVs and a decreased amount of protein cargo within them. In turn, in astrocytes treated with the stress hormone corticosterone, we found an increase of SUMO-2 conjugated proteins and sEVs from these cells contained an augmented protein cargo. In this case, the proteins detected with mass-spectrometry were mostly proteins related to protein translation. To test whether astrocyte-derived sEVs obtained in these experimental conditions could modulate protein synthesis in target cells, we incubated primary neurons with astrocyte-derived sEVs. sEVs from corticosterone-treated astrocytes stimulated protein synthesis while no difference was found with sEVs derived from 2-D08-treated astrocytes. Our results show that SUMO conjugation plays a fundamental role in defining the protein cargo of sEVs impacting the physiological function of target cells.

cell biology↗