Atorvastatin enhances neurite outgrowth in cortical neurons in vitro via up-regulating the Akt/mTOR and Akt/GSK-3β signaling pathways.
OBJECTIVE
To investigate whether atorvastatin can promote formation of neurites in cultured cortical neurons and the signaling mechanisms responsible for this effect.


METHODS
Cultured rat cerebral cortical neurons were incubated with atorvastatin (0.05-10 μmol/L) for various lengths of time. For pharmacological experiments, inhibitors were added 30 min prior to addition of atorvastatin. Control cultures received a similar amount of DMSO. Following the treatment period, phase-contrast digital images were taken. Digital images of neurons were analyzed for total neurite branch length (TNBL), neurite number, terminal branch number, and soma area by SPOT Advanced Imaging software. After incubation with atorvastatin for 48 h, the levels of phosphorylated 3-phosphoinoside-dependent protein kinase-1 (PDK1), phospho-Akt, phosphorylated mammalian target of rapamycin (mTOR), phosphorylated 4E-binding protein 1 (4E-BP1), p70S6 kinase (p70S6K), and glycogen synthase kinase-3β (GSK-3β) in the cortical neurons were evaluated using Western blotting analyses.


RESULTS
Atorvastatin (0.05-10 μmol/L) resulted in dose-dependent increase in neurite number and length in these neurons. Pretreatment of the cortical neurons with phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 (30 μmol/L) and wortmannin (5 μmol/L), Akt inhibitor tricribine (1 μmol/L) or mTOR inhibitor rapamycin (100 nmol/L) blocked the atorvastatin-induced increase in neurite outgrowth, suggesting that atorvastatin promoted neurite outgrowth via activating the PI3K/Akt/mTOR signaling pathway. Atorvastatin (10 μmol/L) significantly increased the levels of phosphorylated PDK1, Akt and mTOR in the cortical neurons, which were prevented by LY294002 (30 μmol/L). Moreover, atorvastatin (10 μmol/L) stimulated the phosphorylation of 4E-BP1 and p70S6K, the substrates of mTOR, in the cortical neurons. In addition, atorvastatin (10 μmol/L) significantly increased the phosphorylated GSK-3β level in the cortical neurons, which was prevented by both LY294002 and tricribine.


CONCLUSIONS
These results suggest that activation of both the PI3K/Akt/mTOR and Akt/GSK-3β signaling pathways is responsible for the atorvastatin-induced neurite outgrowth in cultured cortical neurons.
Department of Pharmacology, Liaoning Medical University, Jinzhou, China. jyjinying@yahoo.com.cn
Ying
Y
Jin
Hai-juan
HJ
Sui
Yan
Y
Dong
Qi
Q
Ding
Wen-hui
WH
Qu
Sheng-xue
SX
Yu
Ying-xin
YX
Jin
D000818:Animals; D000069059:Atorvastatin; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D004789:Enzyme Activation / Q000187:drug effects; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006538:Heptanoic Acids / Q000494:pharmacology*; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology*; D016501:Neurites / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D011758:Pyrroles / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*
D000069059:Atorvastatin; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D006538:Heptanoic Acids; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D011758:Pyrroles; D058570:TOR Serine-Threonine Kinases; C000605508:Gsk3b protein, rat