DJ-1 interacts with and regulates paraoxonase-2, an enzyme critical for neuronal survival in response to oxidative stress.

Loss-of-function mutations in DJ-1 (PARK7) gene account for about 1% of all familial Parkinson's disease (PD). While its physiological function(s) are not completely clear, DJ-1 protects neurons against oxidative stress in both in vitro and in vivo models of PD. The molecular mechanism(s) through which DJ-1 alleviates oxidative stress-mediated damage remains elusive. In this study, we identified Paraoxonase-2 (PON2) as an interacting target of DJ-1. PON2 activity is elevated in response to oxidative stress and DJ-1 is crucial for this response. Importantly, we showed that PON2 deficiency hypersensitizes neurons to oxidative stress induced by MPP+ (1-methyl-4-phenylpyridinium). Conversely, over-expression of PON2 protects neurons in this death paradigm. Interestingly, PON2 effectively rescues DJ-1 deficiency-mediated hypersensitivity to oxidative stress. Taken together, our data suggest a model by which DJ-1 exerts its antioxidant activities, at least partly through regulation of PON2

Regulation of the VHL/HIF-1 Pathway by DJ-1

DJ-1 (PARK7) is a gene linked to autosomal recessive Parkinson disease (PD). We showed previously that DJ-1 loss sensitizes neurons in models of PD and stroke. However, the biochemical mechanisms underlying this protective role are not completely clear. Here, we identify Von Hippel Lindau (VHL) protein as a critical DJ-1-interacting protein. We provide evidence that DJ-1 negatively regulates VHL ubiquitination activity of the α-subunit of hypoxia-inducible factor-1 (HIF-1α) by inhibiting HIF-VHL interaction. Consistent with this observation, DJ-1 deficiency leads to lowered HIF-1α levels in models of both hypoxia and oxidative stress, two stresses known to stabilize HIF-1α. We also demonstrate that HIF-1α accumulation rescues DJ-1-deficient neurons against 1-methyl-4-phenylpyridinium-induced toxicity. Interestingly, lymphoblast cells extracted from DJ-1-related PD patients show impaired HIF-1α stabilization when compared with normal individuals, indicating that the DJ-1-VHL link may also be relevant to a human context. Together, our findings delineate a model by which DJ-1 mediates neuronal survival by regulation of the VHL-HIF-1α pathway

Cdk5-mediated JIP1 phosphorylation regulates axonal outgrowth through Notch1 inhibition

Abstract Background Activated Cdk5 regulates a number of processes during nervous system formation, including neuronal differentiation, growth cone stabilization, and axonal growth. Cdk5 phosphorylates its downstream substrates located in axonal growth cones, where the highly expressed c-Jun N-terminal kinase (JNK)-interacting protein1 (JIP1) has been implicated as another important regulator of axonal growth. In addition, stringent control of the level of intracellular domain of Notch1 (Notch1-IC) plays a regulatory role in axonal outgrowth during neuronal differentiation. However, whether Cdk5-JIP1-Notch1 cooperate to regulate axonal outgrowth, and the mechanism of such joint contribution to this pathway, is presently unknown, and here we explore their potential interaction. Results Our interactome screen identified JIP1 as an interactor of p35, a Cdk5 activator, and we sought to explore the relationship between Cdk5 and JIP1 on the regulation of axonal outgrowth. We demonstrate that JIP1 phosphorylated by Cdk5 at Thr205 enhances axonal outgrowth and a phosphomimic JIP1 rescues the axonal outgrowth defects in JIP1−/− and p35−/− neurons. Axonal outgrowth defects caused by the specific increase of Notch1 in JIP1−/− neurons are rescued by Numb-mediated inhibition of Notch1. Finally, we demonstrate that Cdk5 phosphorylation of JIP1 further amplifies the phosphorylation status of yet another Cdk5 substrate E3-ubiquitin ligase Itch, resulting in increased Notch1 ubiquitination. Conclusions Our findings identify a potentially critical signaling axis involving Cdk5-JIP1-Itch-Notch1, which plays an important role in the regulation of CNS development. Future investigation into the way this pathway integrates with additional pathways regulating axonal growth will further our knowledge of normal central nervous system development and pathological conditions

DJ-1 interacts with and regulates paraoxonase-2, an enzyme critical for neuronal survival in response to oxidative stress.

Loss-of-function mutations in DJ-1 (PARK7) gene account for about 1% of all familial Parkinson's disease (PD). While its physiological function(s) are not completely clear, DJ-1 protects neurons against oxidative stress in both in vitro and in vivo models of PD. The molecular mechanism(s) through which DJ-1 alleviates oxidative stress-mediated damage remains elusive. In this study, we identified Paraoxonase-2 (PON2) as an interacting target of DJ-1. PON2 activity is elevated in response to oxidative stress and DJ-1 is crucial for this response. Importantly, we showed that PON2 deficiency hypersensitizes neurons to oxidative stress induced by MPP+ (1-methyl-4-phenylpyridinium). Conversely, over-expression of PON2 protects neurons in this death paradigm. Interestingly, PON2 effectively rescues DJ-1 deficiency-mediated hypersensitivity to oxidative stress. Taken together, our data suggest a model by which DJ-1 exerts its antioxidant activities, at least partly through regulation of PON2

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

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 Interacts with and Regulates Paraoxonase-2, an Enzyme Critical for Neuronal Survival in Response to Oxidative Stress

Loss-of-function mutations in DJ-1 (PARK7) gene account for about 1% of all familial Parkinson's disease (PD). While its physiological function(s) are not completely clear, DJ-1 protects neurons against oxidative stress in both in vitro and in vivo models of PD. The molecular mechanism(s) through which DJ-1 alleviates oxidative stress-mediated damage remains elusive. In this study, we identified Paraoxonase-2 (PON2) as an interacting target of DJ-1. PON2 activity is elevated in response to oxidative stress and DJ-1 is crucial for this response. Importantly, we showed that PON2 deficiency hypersensitizes neurons to oxidative stress induced by MPP+ (1-methyl-4-phenylpyridinium). Conversely, over-expression of PON2 protects neurons in this death paradigm. Interestingly, PON2 effectively rescues DJ-1 deficiency-mediated hypersensitivity to oxidative stress. Taken together, our data suggest a model by which DJ-1 exerts its antioxidant activities, at least partly through regulation of PON2