Abstract
The ability of the nervous system to undergo long-term plasticity is based on changes in cellular and synaptic proteomes. While many studies have explored dynamic alterations in neuronal proteomes during plasticity, there has been less attention paid to the astrocytic counterpart. Indeed, progress in identifying cell type-specific proteomes is limited owing to technical difficulties. Here, we present a cell type-specific metabolic tagging technique for a mammalian coculture model based on the bioorthogonal amino acid azidonorleucine and the mutated Mus musculus methionyl-tRNA synthetaseL274G enabling azidonorleucine introduction into de novo synthesized proteins. Azidonorleucine incorporation resulted in cell type-specific protein labeling and retained neuronal or astrocytic cell viability. Furthermore, we were able to label astrocytic de novo synthesized proteins and identified both Connexin-43 and 60S ribosomal protein L10a upregulated upon treatment with Brain-derived neurotrophic factor in astrocytes of a neuron-glia coculture. Taken together, we demonstrate the successful dissociation of astrocytic from neuronal proteomes by cell type-specific metabolic labeling offering new possibilities for the analyses of cell type-specific proteome dynamics.
Highlights
Dynamic adaptations in synaptic strength are thought to be the basis for information processing and memory consolidation in neuronal circuits
Dynamic adaptation of the proteome to changes in the cellular environment is one of the universal hallmarks for cellular function and is the basis for a successful collaboration of cells within a complex heterogeneous cellular system. This holds true for the nervous system, where long-term adaptation of neuronal synapses depends on protein synthesis and a new set of proteins might support the stabilization of potentiated synapses [34]
To analyze protein translation events solely in astrocytes that are tightly connected with neurons is technically extremely challenging, since state of the art proteomic approaches demand a separation of both cells to differentiate between their proteomes
Summary
Dynamic adaptations in synaptic strength are thought to be the basis for information processing and memory consolidation in neuronal circuits. In order to fully understand how different modulatory inputs result in plasticity, another major cell type of the central nervous system has to be taken into account, the astrocyte. The intensive crosstalk between neurons and astrocytes at synapses led to the concept of the tripartite synapse model that incorporates modulatory influences on synaptic transmission emanating from astrocytes [1]. Given the close partnership between neurons and astrocytes, it is clear that adaptive mechanisms must exist in the astrocytic part of the tripartite synapse. Adaptive mechanisms, based on astrocytic process dynamics, have been observed in the hypothalamus of lactating animals [3]
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