FIGURE 2.

MPTP up-regulates the levels of ER chaperones and ERAD component by activating ATF6α and induces the phosphorylation of p38 MAPK in dopaminergic neurons in vivo. A–H, extracts of the midbrain or the striatum of ATF6α WT(+/+) and ATF6α KO(−/−) mice were prepared 5 days after intraperitoneal injection with either 20 mg/kg MPTP or normal saline four times every 2 h for Western blot analysis of ATF6α, PERK, p38MAPK, phospho-PERK (p-PERK), phospho-p38MAPK (p-p38MAPK), BiP, GRP94, Derlin-3, and β-actin (n = 4 for each group). The protein levels of p-PERK and p-p38MAPK were quantified using optical density and estimated relative to PERK or p38MAPK protein level, respectively. Other protein levels were estimated relative to β-actin protein level. Each bar denotes the mean ± S.D. MPTP induces ATF6α cleavage (A and C), phosphorylation of PERK (A and D) and p38MAPK activation (A and E) in the midbrain. MPTP up-regulates the levels of BiP (B and F), GRP94 (B and G), and Derlin-3 (B and H) in the striatum in vivo. Abbreviation: N.S. indicates normal saline injection as control. I–L, MPTP up-regulates the levels of BiP and p-p38MAPK in TH-positive neurons. The midbrains of ATF6α WT mice treated with MPTP or normal saline (Control) were stained using anti-TH (I–L, green), BiP (I and J, red), p-p38MAPK (K and L, red) antibodies, and DAPI (K and L, blue). They were visualized by Alexa Fluor 488- and Alexa Fluor 546-conjugated secondary antibodies and subjected to confocal fluorescence microscope analysis. White arrowheads in J and L indicate the TH-positive dopaminergic cells of MPTP-treated mice. Scale bars indicate 10 μm.