Figure 6.

miR-7 and/or miR-153 attenuated MPP⁺-induced neurotoxicity via upregulation of rapamycin sensitive mTOR pathway. (A,B) Six to seven days old primary cortical neurons were transduced with adenoviral particles expressing scramble miR, miR-7, miR-153, or both miR-7/153. After 24 h, transduced neurons were exposed for additional 24 h to 10 μM of MPP⁺. Equal amounts of total protein from lysates of transduced cortical neurons cultured for 24 h in the presence of 10 μM MPP⁺ were analyzed on 10% SDS-PAGE and immunoblotted with antibodies specific for phosphorylated forms of p70S6K (A) and S6RP (B). To ensure equal loading membranes were re-probed against p70S6K and S6RP, respectively. Note that overexpression of miR-7 and/or miR-153 attenuated the effect of MPP⁺ on the phosphorylation of all proteins examined. Quantification of the results was performed by scanning densitometry. (C) Six to seven days old cultures of primary cortical neurons were transduced as described above, and 24 h later were exposed to 20 nM of rapamycin, 10 μM of MPP⁺, or both. Neuronal viability was monitored 24 h post-exposure by the MTT assay. Note that in the presence of rapamycin, overexpression of miR-7 and/or miR-153 failed to protect cortical neurons against MPP⁺-induced cell death. Bars in all presented graphs depict mean ± s.e.m. ^*P < 0.05, ^***P ≤ 0.001.