Fig 2: Effects of JNK3-N-Tat on the downstream effectors of JNK3 in PD models.(A) Western blotting analysis of p-c-Jun, T-c-Jun and cytochrome c in SH-SY5Y cell extracts after treatment. The expression levels of cytochrome c and p-c-Jun in the JNK3-N-Tat-treated groups were clearly decreased compared with the MPP+- and JNK3-4A-Tat-treated groups. (C) Cytosolic and nuclear fractions were separated, and nuclear and cytosolic T-JNK3 levels were assessed by Western blotting. The results revealed decreased nuclear translocation of T-JNK3 in the JNK3-N-Tat group compared with the vehicle group. Treatment with JNK3-N-Tat did not result in JNK3 returning to the cytoplasm. (E) Western blotting analysis of p-c-Jun, T-c-Jun, cytochrome c and TH in extracts from mouse SNc. The expression of TH in JNK3-N-Tat-treated mice was greater than in MPTP- or JNK3-4A-Tat-treated mice. JNK3-N-Tat treatment also attenuated the MPP+-induced activation of c-Jun and cytochrome c in the MPTP mouse model (n = 6 animals per group). (B, D, F) Bar graphs display the results of our statistical analysis (mean±SD). NC: Normal control; N-Tat: JNK3-N-Tat; 4A-Tat: Tat-4A-Tat. a: P<0.05 versus Normal control; b: P<0.05 versus vehicle; c: P<0.05 versus JNK3-N-Tat; d: P>0.05 versus vehicle; #: P>0.05 versus JNK3-N-Tat.

Fig 3: Effects of JNK3-N-Tat on mitochondrial damage and apoptosis in MPP+-treated primary cultured cortical neurons.Representative images of the sham-treated normal control group (i) and the groups administered saline vehicle (ii), JNK3-N-Tat (iii), or JNK3-4A-Tat (iv) before MPP+ treatment to cortical neurons. Pretreatment with 20 µm JNK3-N-Tat or JNK3-4A-Tat was performed 1 hr before MPP+ treatment (100 µM). The cells were then cultured for 6 hr, followed by fixation or staining with donkey anti-T-JNK3 and donkey anti-ß-arrestin2 antibodies. (A) Co-localization of T-JNK3 and ß-arrestin2 in MPP+-treated cortical neurons. Representative confocal images show that JNK3-N-Tat inhibited the co-localization of JNK3 and ß-arrestin2 induced by MPP+ in primary cultured cortical neurons. (B) Electron microscopy revealed that JNK3-N-Tat treatment protected neuronal mitochondria against MPP+-induced injury. (C, D) Flow cytometry revealed that JNK3-N-Tat treatment attenuated the MPP+-induced neuronal apoptosis. Bar graphs show the results of our statistical analysis (mean±SD) from three independent experiments. a: P<0.05 versus Normal control; b: P<0.05 versus vehicle; c: P<0.05 versus JNK3-N-Tat; d: P>0.05 versus vehicle.

Fig 4: JNK3-N-Tat-Dansyl, but not Tat38-48-Dansyl, accumulates within neurons.(A) SH-SY5Y cells were treated with JNK3-N-Tat-Dansyl (a-i), Tat38-48-Dansyl (j) or vehicle (k) for the indicated periods. JNK3-N-Tat-Dansyl (20 µM), but not Tat38-48-Dansyl (20 µM), exhibited strong fluorescence under UV light 30 min after application to SH-SY5Y cell cultures (40× magnification). The arrows indicate co-localization of JNK3-N-Tat-Dansyl with the SH-SY5Y cell body. WL: white light. (B) Fluorescence was detected under UV light in coronal sections from the SNc (Fig. B, a and c) of mice injected i.p. with JNK3-N-Tat-Dansyl (1 µg/g.kw). Arrows indicate the localization of JNK3-N-Tat-Dansyl in the SNc. n = 3. Scale bars in B (a–d): 1 = 20 µm.

Fig 5: Activation of the ASK1-JNK3 signaling pathway and binding of ß-arrestin2 to JNK3 were increased in MPP+-treated cells.SH-SY5Y cells were treated with MPP+ (3 mM) or vehicle for the indicated times. (A, C) Western blotting analysis of p-ASK1, T-ASK1, p-c-Jun and T-c-Jun in extracts from SH-SY5Y cells after MPP+ treatment. (E) p-JNK3 was examined using IP and IB analyses in lysates from SH-SY5Y cells treated with MPP+ for 12 hr. The results revealed that p-JNK3 expression was increased and that the binding of ß-arrestin2 to JNK3 was significantly increased. (G, I) Neurons were treated with MPP+ (100 µM) or vehicle for the indicated times. Western blotting analysis of p-ASK1, T-ASK1, p-c-Jun and T-c-Jun in extracts from cells after MPP+ treatment. (B, D, F, H, J) Bar graphs show the results of our statistical analysis (mean±SD) from three independent experiments. Flow: Flow-through; Input: Supernatant of non-denaturing lysis buffer. * P<0.05; ** P<0.01; # P>0.05.