FIG. 6.

Decreased DA cell death by Nox1 knockdown or Rac1 inhibition. (A and B) N27 cells were incubated with Nox1 shRNA/ AAV particles for 36 h and then exposed to 6-OHDA (100 μM) for 6 h. Cultures were stained with phospho-c-Jun (p-c-Jun, red). Cells expressing GFP (green) represent AAV-transduced cells. DAPI staining (blue) was used to visualize nucleus (A). GFP-positive and p-c-Jun-negative cells (p-c-Jun-, green) and GFP-positive and p-c-Jun-positive cells (p-c-Jun+, yellow) cells were counted. Total 726 GFP-positive cells were counted. Data represent three independent experiments with similar results. ***p<0.001 (B). (C and D) Representative photomicrographs of TH staining in the rat SN sections. AAV particles containing empty vector, scramble shRNA, Nox1 shRNA, or T17NRac1 were stereotaxically injected into the rat SN. After 4 weeks incubation, vehicle or 6-OHDA were injected into the striatum. Two weeks later, DA neurons in the SN were visualized with TH immunostaining (upper panel) for T17N Rac1. For Nox1 shRNA, the SN was stained both TH and Nissl. Vector, injection of vector/AAV particles into the SN; T17N Rac1, injection of T17NRac1/AAV particles into the SN; scb shRNA, injection of scramble shRNA/AAV into the SN; Nox1 shRNA, injection of Nox1 shRNA/AAV particles into the SN;+vehicle, striatal injection of vehicle 4 weeks after AAV;+6-OHDA, striatal injection of 6-OHDA 4 weeks after AAV. Scale bar=150 μm (C). Stereologic counts of TH-positive neurons in the SN shown as percentage of vector+vehicle or scb shRNA+vehicle (left and middle panel). Nissle-positive neurons in the SN shown as percentage of scb shRNA+vehicle. Results are presented as the mean+SEM. n=6–7. Significance is indicated by ***p<0.001 and **p<0.01 (D). (E) p-c-Jun was also detected in the SN by immunohistochemistry. Scale bar=100 μm.