Reduction of O1 and MBP expression by pharmacological inhibition of the tmAC.

SC-neuron cultures were induced to produce myelin by treatment with ascorbate in the absence (Control) or presence of SQ22536 (SQ), which was provided at 0.8 μM unless otherwise indicated. Treatment was carried out for 12 days (A-B) and 5 days (D), respectively. Cultures were analyzed for their expression of O1 and MBP (A-B) or O1 and P-PKA-specific substrates (D) by immunofluorescence microscopy. A quantification of O1 and MBP expression is provided in B, which also includes a condition where mtAC activity was inhibited by dideoxy-adenosine (ddA). As shown in A, some SCs displayed high levels of cell surface O1 even in the presence of SQ22536. However, these cells did not convert into myelinating cells, as denoted by their multiple processes (indicative of failure to establish a one-to-one association with axons) and lack of MBP expression. The dose dependency of P-PKA substrate expression by increasing concentrations of SQ22536 was confirmed by western blot analysis in SC-only cultures (C). The arrowheads in B indicate a source of P-PKA substrate immunoreactivity exclusively present in differentiating O1 positive cells (boxes) that was insensitive to inhibition by SQ22536.