Fig. 8. ASK1 activation contributes to nigrostriatal neurodegeneration in 6-OHDA-lesioned mice.

(A) Lentivirus-mediated knockdown of ASK1 expression in mouse SNc. Mouse SNc was infected for 21 days with empty lentiviral vector, Lenti-ASK1t, or Lenti-ASK1sc, and then subjected to immunoblotting against ASK1 and double-label immunofluorescent staining for ASK1 and TH. ***P<0.001 vs. empty vector control; n=5/group. Scale bars: 50 µm for low magnification (a–f); 20 µm for high magnification (g–l). (B–C) ASK1 knockdown attenuates the ASK1 signaling cascade in 6-OHDA-lesioned mice. Mouse SNc was infected for 21 days with the lentiviral vectors described in (A), and the mice received striatal infusion of 6-OHDA (3.0 µg) or saline. Immunoblotting against p-ASK1 and its downstream signaling proteins was performed 7 days after 6-OHDA infusion (B); quantitative data of (B) are illustrated in (C). *P<0.05; **P<0.01 vs. vehicle control; #P<0.05; ##P<0.01 vs. empty lentivirus group, n=5–6/group. (D–G) ASK1 knockdown attenuates DA neuron loss and neurological deficits in 6-OHDA-lesioned mice. Mice were treated as described in (A); behavioral tests (D, E) and histology (F, G) were performed 21 days after 6-OHDA infusion. Apomorphine-induced circling after 6-OHDA injection is shown as the number of turns in the first 30 min after intraperitoneal injection of apomorphine (D). The corner test shows the mean of the ratio of spontaneous left/right turns out of 10 trials/session 21 days after 6-OHDA injection (E). Arrows in (F) indicate the SNc region lesioned by 6-OHDA. The numbers of TH-positive neurons in the SNc of 3 experimental groups are illustrated in (G). (H) Catecholamine levels from the ipsilateral striatum 21 days following the indicated injections. Catecholamines are expressed as picomoles/mg of fresh, wet tissue. *P<0.05; **P<0.01 between the groups indicated. n=10–12 per group for all experiments.