Fig. 4.

Modulation of autophagy increases myelin production in vitro. Immunoblots showing the ability of autophagy inhibitor compound CC (2.5 µM) to inhibit autophagy without affecting the normal autophagic flux (A) and to restore normal myelin levels following treatment with TNF-α or IL-1-β, either alone or in combination (B). Early-stage autophagy inhibitor 3-MA (2.5 µM) reduces autophagic activities, as suggested by a decrease in LC3-II levels (C) and reverts TNFα- or IL-1-β–mediated loss of myelin (D). Genetic interference on the essential autophagic gene ATG7 decreases the autophagosomal formation (E) and restores the myelin loss provoked by proinflammatory cytokines (F). The late-stage inhibitor CQ blocks autophagy by interfering with LC3-II degradation. At demonstration of this, the vacuolar H⁺-ATPase inhibitor BafA1 that inhibits the autophagosome–lysosome fusion does not increase LC3-II levels than the CQ (1 µM) treatment alone (G). Interestingly, clozapine (Cloz) and haloperidol (Halo) 1 µM exert the same effect as CQ (H and I). Clozapine and haloperidol (like the late-stage autophagy inhibitor CQ) also revert TNFα- or IL-1-β–mediated loss of myelin (J–L). All experiments are performed in MG cultures. Immunoblots are representative of at least three independent experiments. Quantification of the Western blot performed is reported in SI Appendix, Table S1.