Protein Synthesis in Astrocytes: 'Spontaneous' and Cyclic AMP-Induced Differentiation

Abstract

Primary cultures of mouse astrocytes have been used to study astroglial protein synthesis during ''in vitro'' differentiation. Spontaneous age-related differentiation was compared to the effect of DBcAMP or forskolin, a drug which directly stimulates the adenylate cyclase and induces ''morphological differentiation'' in these cells. Cell differentiation was followed in parallel by phase contrast microscopy and immunofluorescence techniques. Two antisera, one raised against GFA, the other against microtubule-associated protein 2 (MPA2) were used. Anti-GFA serum labelled the cells as early as 7 days in vitro. Anti-MAP2 serum revealed a dense fibrous network at later stages of the culture, whereas the dividing astroblasts appeared poorly stained by this antibody. Both phase contrast microscopy and immunofluorescence techniques suggested that most of the cells spontaneously differentiate after 3 weeks of culture even in the absence of DBcAMP for forskolin. Forskolin, while accelerating differentiation after 7 days of culture, produced smaller cells than DBcAMP and had biphasic effects on cell morphology. Mono- and two-dimensional gel electrophoresis of the 35S-methionine labelled cells also showed that the major changes in protein synthetic activity occur spontaneously during the time course of the culture. Whatever the stage of the culture, DBcAMP for forskolin induced changes in the synthesis of only a few proteins. However, depending on the culture stage the proteins, which were positively or negatively controlled by these drugs, were not the same.