Figure 4.

Knockdown of ArfGAP1 expression reduces LRRK2 toxicity. A, Total cell lysates from NIH 3T3 cells transduced by GFP-tagged LV.shRNA GAPDH, LV.shRNA ArfGAP1-1, LV.shRNA ArfGAP1-2, and LV.shRNA ArfGAP1-3 for 5 d were subjected to immunoblotting with anti-ArfGAP1 and anti-tubulin antibodies. B, Quantification of ArfGAP1 levels normalized (nanograms) to the percentage of cells treated with LV.shRNA GAPDH. Data are representative of three independent experiments. Bars represent the mean ± SEM. Data were analyzed for statistical significance by Student's t test (*p < 0.05). LV.shRNA–ArfGAP1-2 was chosen for future studies. C, Representative fluorescent images (red) showing mouse primary cortical neurons transduced with GFP-tagged LV.shRNA GAPDH or LV.shRNA ArfGAP1 for 3 d (>95% neurons are GFP positive) before cotransfection with LRRK2 and mCherry at a 10:1 molar ratio. Neuronal viability was analyzed 48 h after transfection (DIV 12) with nonviable neurons exhibiting no obvious neurite process (arrow) (GS, G2019S; DA, D1994A). D, Quantification of neuronal viability. Bars indicate the viability of mCherry-positive neurons (n = 200) for each transfection condition expressed as a percentage of control neurons (mCherry with pcDNA3.1 empty vector). Data represent the mean ± SEM from three independent experiments. Data were analyzed for statistical significance by the Student's t test (*p < 0.05). E, Exogenous LRRK2 expression levels were not significantly changed with downregulation of ArfGAP1. Cell lysates were collected from primary cortical neurons transduced with LV.shRNA GAPDH or LV.shRNA ArfGAP1 for 3 d before cotransfection with LRRK2 and mCherry at a 10:1 molar ratio for 48 h. Cell lysates were subjected to immunoblotting with anti-Myc or with anti-actin to show an equivalent amount of input. Data represent the mean ± SEM from three independent experiments. ns, Nonsignificant. F, Exogenous LRRK2 cellular localization was not significantly changed with downregulation of ArfGAP1. Primary cortical neurons transduced with LV.shRNA GAPDH or LV.shRNA ArfGAP1 for 3 d before cotransfection with LRRK2 and mCherry at a 10:1 molar ratio for 48 h were stained with anti-Myc and anti-GFP antibodies, followed by detection of secondary antibodies conjugated to Alexa Fluor-488 (GFP, green) and Alexa Fluor-405 (LRRK2, blue). Enlarged images (highlighted by the white dashed box in the left LRRK2 panels) for exogenous LRRK2 staining are shown on the right panel for LRRK2. Scale bars: 20 μm; high-power images, 5 μm. G, No significant difference was observed on ArfGAP1 toxicity in WT and LRRK2 KO neurons. Representative high-power fluorescent images (GFP) showing WT and LRRK2 KO mouse primary cortical neurons transfected with ArfGAP1–GFP. Neuronal viability was analyzed at 48 h after transfection (DIV 12) with nonviable neurons exhibiting no obvious neurite process and positive TUNEL staining (arrow). H, Quantification of neuronal viability. Bars indicate the viability of GFP-positive neurons (n = 200) for each transfection condition expressed as a percentage of control neurons cotransfected with GFP alone. Data represent the mean ± SEM from three independent experiments. WB, Western blot.