In this study we find that (1) Methylene blue protects HT22 cells from serum deprivation-induced apoptosis through induction of macroautophagy; (2) Methylene blue induces macroautophagy via enhancement of AMPK pathway rather than inhibition of mTOR pathway in vitro; (3) Methylene blue induces macroautophagy via enhancement of AMPK signal pathway in mouse hippocampus and cortex in vivo; (4) Methylene blue does not induce macroautophagy in neural stem/progenitor cells located at the mouse SVZ in vivo.
Neurons in central nervous system are sensitive to inadequate serum supply. Studies have demonstrated the morphological and metabolic characteristics of neurons after serum deprivation (LeBlanc, 1995
; Bae et al., 2011
; Lesort et al., 1997
; Poser et al., 2003
; Chauvier et al., 2005
; Steiger-Barraissoul and Rami, 2009
). In the current study, we extend our previous findings and demonstrated that methylene blue exerts neuroprotective effect against serum deprivation. Recentstudies suggest that macroautophagy is neuroprotective against apoptosis induced by serum deprivation (Steiger-Barraissoul and Rami, 2009
). Under nutrient starvation or energy deprivation circumstances, macroautophagy is thought to protect cells via saving and recycling materials for maintaining the synthesis of vital macromolecules. Our data showed that methylene blue treatment effectively protected starved cells from apoptosis and induced macroautophagy. When macroautophagy was blocked by chloroquine, the methylene blue-mediated neuroprotection was partially inhibited, suggesting that macroautophagy is involved in the anti-apoptotic action of methylene blue.
Previous studies have indicated that mTOR-related signal transduction is the key regulator of macroautophagy (Levine and Kroemer, 2008
; Rabinowitz and White, 2010
). Other signal pathways such as Akt and MAPK/Erk1/2 influence macroautophagy indirectly through regulating mTOR pathway. A very recent study has suggested that methylene blue induces macroautophagy to attenuate tauopathy through inhibiting mTOR activation (Congdon et al., 2012
). Our data demonstrated that methylene blue did not significantly activate mTOR signaling both in vitro
and in vivo
. Consistently, Akt and Erk signaling, which has been proven to activate mTOR pathway, were not inhibited after methylene blue treatment. Bcl-2 was suggested to inhibit Beclin1-dependent macroautophagy. Methylene blue robustly increased Bcl-2 protein levels. Therefore, we speculated that Bcl-2 is not involved in methylene blue-induced macroautophagy. Rather, the anti-apoptotic Bcl-2 might be another mechanism contributes to the neuroprotectiove action of methylene blue.
5′ Adenosine monophosphate-activated protein kinase plays as a master regulator of cellular energy homeostasis (Hardie et al., 2012
). The kinase is activated in response to stresses that deplete cellular adenosine triphosphate (ATP) supplies such as low glucose, hypoxia, ischemia, and heat shock. AMPK activation positively regulates signal pathways that replenish cellular ATP supplies while negatively regulates several proteins central to ATP consuming processes. It has been demonstrated that AMPK signaling positively regulate macroautophagy by activating Ulk1 through phosphorylation of Ser317 and Ser777, or indirectly by inhibiting mTOR signaling (Mihaylova and Shaw, 2011
). Our data indicated that methylene blue induces macroautophagy via, at partially, AMPK signaling. Both AMPKα and AMPKβ1/2 protein levels were increased in vitro
, whereas the changing pattern was more complicated in vivo
, depending on the brain regions. In the hippocampus and cortex, the action of methylene blue on macroautophagy was similar to that in vitro
, while no effect of methylene blue was observed in SVZ cells. Since SVZ consists of primitive neural stem/progenitor cells, we speculated that methylene blue-induced AMPK activation mainly occurs in mature neurons. Elaborating studies have shown that methylene blue act as an alternative electron carrier in the mitochondrial electron transport chain to enhance ATP production while energy is in short. So it is unlikely that AMPK activation in methylene blue-treated cells was due to increased AMP/ATP ratio. Promoted ACC phosphorylation indicated AMPK pathway was indeed activated. Methylene blue-induced AMPK activation was accompanied by higher levels of LC3B type II, the activated form of LC3 participating autophagosome formation. Furthermore, when AMPK inhibitor was administrated with methylene blue, ACC phosphorylation was reverted to basal level together with reduced LC3B type II level.
Taken together, the present study suggests that methylene blue exert protective effect against serum deprivation, at least partly, through enhancing macroautophagy. In addition, our study demonstrated that activation of AMPK pathway, but not mTOR, is involved in the methylene blue-induced macroautophagy.