Fig. 3.

Pyridazine analogs induce EAAT2 locally translation in gliosomes. Gliosomes were isolated from untreated mice and vehicle or compound was applied directly to gliosomes ex vivo as depicted. (A, B) Western blot analysis showed that active pyridazine compounds induce de novo synthesis of EAAT2 in ex vivo gliosomes in a dose- and time-dependent manner. (C) However, inactive pyridazine analogs do not induce the local translation of EAAT2 in gliosomes. (D) Ex vivo induction of EAAT2 resulted in insertion of EAAT2 into the plasma membrane. Pulldown of biotinylated extracellular proteins after ex vivo treatment showed increased EAAT2 insertion in line with increased total expression (E) In support of the feasibility for local translation to occur, RT–PCR analysis showed expression of the full-length EAAT2 mRNA in gliosomes, but not in the synaptosome fraction, and the 18S ribosomal RNA in all fractions. This suggests that the major components necessary for translation exist in gliosomes. Each study consisted of four replicates with consistent results. Representative blots are shown.