METHYLENE BLUE INHIBITS PROLIFERATION AND MAINTAINS SELF-RENEWAL OF RAT NEURAL STEM/PROGENITOR CELLS

Neural progenitor cells (NPCs)are important for neurogenesis and brain damage repair. To elucidate the effect of methylene blue on NPCs, we studied the self-renewal and proliferation of NPCs both in vitro and in vivo. We found that methylene blue restrains NPC proliferation and enahnces NPC self-renewal. Purpose (a): Neural stem cell-based treatment holds a new therapeutic opportunity for treating neurodegenerative disorders. While methylene blue has been shown to be neuroprotective in multiple experimental neurodegenerative disease models, its potential effects on neural stem/progenitor cells (NSPCs) has not been addressed. Methylene blue can easily penetrate the blood brain barrier to access the brain parenchyma. Thus, its effects on NSPCs, whether positive or negative, need to be elucidated. Methods (b): We used in both in vitro culture model and in vivo study to test the effects of methylene blue on the proliferation, self-renewal and differentiation of NSPCs. Neurospheres were generated in vitro and were treated with methylene blue. NSPC proliferation was evaluated by Ki67 staining and propidium iodide staining. NSPC self-renewal was determined by serial passage assay. Real-time PCR was applied to test the expression of neural differentiation markers in NSPCs. To address the signal pathway responsible for the methylene blue-induced changes on NSPCs, expression of cyclins and mTORC1 activation were determined by real-time PCR and Western blot, respectively. The effects of methylene blue on NSPC proliferation was also confirmed by intracerebroventricular infusion of methylene blue in rats followed by BrdU and Nestin staining. Results (c): Methylene blue inhibits porliferation of rat NSPCs in the in vitro culture. Methylene blue treatment decreased most cyclin expression. In addition, methylene blue enhanced the self-renewal capacity of NSPCs, demonstrated by more neurosphere growth and inhibited differentiation marker expression in NSPCs. However, methylene blue did not impair committed neuronal differentiation. The change of cyclin expression is associated with the change of mTOR expression in methylene blue-treated NSPCs. Methylene blue repressed transcription of mTOR rather than enhance mTOR degradation. Consistent with in vitro data, methylene blue inhibited neural stem cell division in the subventricular zone, but did not influenced neuronal development in a short term. Conclusions (d): Our findings indicate that methylene blue could delay NSPCs senescence by enhancing NSPCs self-renewal capacity. However, the long-term effects of methylene blue on the in vivo NSPC pool needs further investigation. The impact of methylene blue on NSPCs should be taken into account in future therapy with methylene blue, either for the peripheral diseases or for the CNS disorders.