PON2 protects neurons against MPP^<b>+</b>.

<p>(<b>A</b>) Primary cortical neurons obtained from PON2 deficient or wild type mice were subjected to 10, 20 and 40 µM MPP⁺ treatment for 48 hours. Cells were lysed and viability was assessed by direct microscopy and counting intact nuclei. (<b>B</b>) WT and PON2 def cortical neurons were transfected with plasmid expressing Myc-PON2 and GFP (under independent promoters), or GFP as control, and subjected to 20 µM MPP⁺ for 48 hours. Cells were fixed and the nuclei were stained with Hoechst. Survival percentage represents the ratio of GFP-expressing cells with morphologically intact nuclei (D, a and b) to the total number of GFP positive cells. (<b>C</b>) WT and DJ-1 KO cortical neurons over-expressing PON2 and GFP or GFP alone as control (using adenovirus expressing PON2 or GFP) were subjected to 20 µM MPP⁺ for 48 hours. The survival assay was performed as described in part B. (<b>D</b>) Representative image of GFP positive neurons (a and c), and Hoechst-stained surviving (b) and dead (d) nuclei. (<b>E</b>) Western blot analysis of PON2 levels in PON2 deficient (PON2 def) and WT MEFs and also in WT MEFs infected with PON2-expressing adenovirus (WT+PON2 AV). The membrane was probed with PON2 antibody. (<b>F</b>) Western blot analysis for Myc in WT MEFs expressing control (Ctr) or Myc-PON2 plasmids. The Western blot was analyzed by Myc antibody. Statistical significance was assessed by Anova and post-hoc test Tukey on data obtained from three independent experiments (n = 3). * denotes p<0.05, **denotes p<0.01 and *** denotes p<0.001.</p

DJ-1 and oxidative stress modulate PON2 activity.

<p>(<b>A</b>) Cultured WT and DJ-1 KO cortical neurons were treated with MPP⁺ (20 µM) for 12 hours and cells were washed and membrane was extracted. Crude membrane was exposed to the substrate C12 for 60 minutes and the percentage of remaining C12 was measured. (<b>B</b>) Cultured WT and DJ-1 KO cortical neurons were treated with MPP⁺ (20 µM) for 24 hours. Neurons were then exposed to DHC for 10 minutes and the amount of hydrolysis of DHC was assessed with measuring UV absorbance. One unit of PON2 activity is equal to 1 µmol DHC hydrolyzed<b>/</b>ml<b>/</b>min. (<b>C</b>) WT and DJ-1 KO MEFs were treated with hydrogen peroxide (100 µM) for 24 hours and PON2 activity was measured as described in B. (<b>D</b>) WT and DJ-1 KO MEFs were infected with adenovirus expressing DJ-1 or GFP alone as control. After 48 hours of expression, cells were lysed and exposed to C12 as the substrate for 60 minutes. Percentage of C12 remaining in activity buffer was measured. Statistical significance was assessed by Anova and post-hoc test Tukey on data obtained from three independent experiments (n = 3). * denotes p<0.05, ** denotes p<0.01, and *** denotes p<0.001.</p