FIGURE 6.

B3C ameliorates MPTP-induced impairments of MEF2D in an in vivo model of PD. A, co-localization of TH and MEF2D in the SNc region. Midbrain slices were prepared from 14-week-old mice and analyzed by co-immunofluorescence (TH, red; MEF2D, green). Scale bar = 20 μm. B, attenuation of MPTP-induced inhibition of Akt pathway by B3C in C57BL/6 mice. C57BL/6 mice were treated with saline (control (CTL)), MPTP alone, or MPTP/B3C by intraperitoneal injection. The lysates of mouse brain tissues from SNc region collected at day 7 after final injection were analyzed using the indicated antibodies. The levels of protein were quantified as the ratio relative to the control (n = 9; *, p < 0.05, **, p < 0.01). pSer473-Akt, phospho-Ser-473-Akt; pSer9-GSK3β, phospho-Ser9-GSK3β. C, attenuation of MPTP-induced increase in H₂O₂ in mouse brain by B3C. As described in B, the H₂O₂ level in mouse brain tissues homogenate was tested by using the Cayman kit (n = 9; *, p < 0.05). D, B3C does affect basal levels of phospho-Akt and -GSK3β. C57BL/6 mice were treated with saline and B3C by intraperitoneal injection. The lysates from brain SNc region collected at day 7 after injection were analyzed using the indicated antibodies. E, attenuation of MPTP-induced reduction of nuclear MEF2D in mice by B3C. Nuclear fractions were prepared from brain tissues from mice treated as described in B and analyzed for nuclear MEF2D (n = 6; *, p < 0.05). F, attenuation of MPTP-induced decrease in mitochondrial MEF2D. Mitochondria were prepared from midbrain tissues from mice as treated in B and analyzed for mitochondrial MEF2D, ND6, and VDAC. The levels of MEF2D and ND6 were quantified (saline control was set as one; n = 9; *, p < 0.05). G, B3C enhances expression of MEF2D in both nuclei and mitochondria. Nuclear and mitochondrial fractions were prepared from brain SNc region from mice treated as described in A and analyzed for MEF2D and H1 (left panel) and VDAC (right panel).