Figure 5.

Inhibiting ERK1/2 activation restores respiratory complex protein expression and function. A significant decrease of NDUFA9 protein was observed in cells with 250 μM MPP⁺ × 2 weeks, which was prevented by 5 μM U0126 (a, b, n=4). **P<0.01, MPP⁺ versus ctrl, ctrl+U0126, and MPP⁺/U0126. The complex I subunit NDUFB8, complex IV-cytochrome oxidase subunit II and complex III core 2 were also decreased with MPP⁺, and restored by co-treatment with U0126 (c). Mitochondrial oxygen consumption was analyzed using an XF24 Extraceullar Flux Analyzer. Oxygen consumption rate (OCR) curves are shown under basal conditions (d) and after addition of 1 μM oligomycin (D-1), 300 nM FCCP (D-2), 120 mM 2-DG (D-3), and 1 μM rotenone (D-4). MPP⁺ caused significant decrease of basal (before 1) and maximal (between 2 and 3) FCCP-induced OCR. U0126 not only reversed the MPP⁺-induced OCR inhibition, but also increased mitochondrial reserve capacity (RC) beyond that of control cells (e). **P<0.01 MPP⁺ versus Ctrl, ctrl +U0126, and MPP⁺+U0126; ^†P<0.01 MPP⁺+U0126 versus ctrl or ctrl+U0126. Cells were shifted into galactose as a fuel source for 24 h to stimulate dependence on mitochondrial respiration. Under this condition, MPP⁺ elicited a reduction in ATP levels that was reversed by concurrent treatment with U0126. *P<0.05 as indicated (f). RNAi-mediated knockdown of ERK1/2 also diminished the decrease in TFAM, complex I and IV subunits that were elicited by MPP⁺ (250 μM, 2 weeks) (g; Supplementary Figure S3G for densitometry)