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

The Mechanisms of Protective Function of DJ-1 in Parkinson’s Models of Neuronal Loss: VHL and PON2

Parkinson's disease (PD) is the most common neurodegenerative motor disorder, whose clinical features are rest tremor, bradykinesia, muscular rigidity and postural instability. Although most reported cases are sporadic, a handful of familial cases and their causative genes have been identified. Loss-of-function mutations in DJ-1, one of these genes, are responsible for 1% of familial PD cases. Our laboratory has previously reported that DJ-1- lacking neurons are sensitive to oxidative stress, induced by hydrogen peroxide or the neurotoxin MPTP. To investigate the possible mechanisms through which DJ-1 protects against oxidative stress, we performed a proteomic screen and identified Von Hippel Lindau (VHL) and Paraoxonase2 (PON2) as potential DJ-1 interacting partners. VHL is an E3 ubiquitin ligase which, in normal conditions, poly-ubiquitinates HIF-1 , a subunit of a master hypoxic/oxidative stress transcription factor, whose function is protective in oxidative and hypoxic stresses. In the present study, we provided further evidence of interaction of DJ-1 with VHL. We also demonstrated that HIF-1 protein level, as an indicator of VHL activity, is lower in cells lacking DJ-1, suggesting the inhibitory role of DJ-1 on VHL. Our in vitro studies also showed that DJ-1 inhibits ubiquitin ligase activity of VHL on HIF-1 by reducing the VHL-HIF-1 interaction. Importantly, accumulation of HIF-1 protects embryonic cortical neurons against MPP+ induced neuronal death. Finally, we confirmed the impairment of HIF-1 response to oxidative stress in human lymphoblastoids of DJ-1-linked PD cases. In the second part of this study, we demonstrated the interaction of DJ-1 and PON2. Interestingly, PON2 lactonase activity is reduced in DJ-1 deficient cells which could be rescued by re-introduction of DJ-1, suggesting a modulating role of DJ-1 on PON2 activity. In addition, PON2 deficiency, like DJ-1 deficiency, hypersensitizes neurons to MPP+, which could be rescued by over-expression of PON2 in both cases. Taken together, our data provide evidence that DJ-1 exerts its protective role by inhibiting VHL activity, enhancing HIF-1 stability, and increasing PON2 pro-survival function in PD models

The Correlation of Brody High Resolution Computed Tomography Scoring System with Clinical Status and Pulmonary Function Test in Patients with Cystic Fibrosis

Background: To reduce the mortality and morbidity rates of cystic fibrosis (CF) patients, and to have an effective clinical management, it is important to monitor the progression of the disease. The aim of this study was to evaluate the progression of lung disease in CF patients by means of assessing the correlation of the CT scoring system with clinical status and pulmonary function test at the Pediatric Pulmonary Ward of Masih Daneshvari Hospital in 2008. Methods: Pulmonary high resolution computed tomography (HRCT) was performed in 23 CF patients using the Brody's scoring system. Morphologic signs as well as the extent and severity of each sign were scored, and the total score was calculated. The correlation of HRCT scores (total score as well as the score for each parameter) with Shwachman Kuczycki scoring system and pulmonary function test were examined. Results: The study included 9 female and 14 male patients with an age range of 5-23 years (mean: 13.42 years). Bronchiectasis (100%) and peribronchial wall thickening (100%) were the most frequent CT abnormalities. Mucus plugging, air trapping and parenchymal involvements were respectively seen in 95.7%, 91.3% and 47.8% of patients. The overall CT score for all patients was 57.6±24.2 (means±SD). The results of pulmonary function test showed a restrictive pattern; however, in 5.3% of the patients PFT was normal. The overall Shwachman-Kulczycki score was 53.48±13.8. There was a significantly (P=0.015) negative correlation between the total CT score and Shwachman-Kulczycki score; however, there was no significant correlation between total CT score and the results of PFT (P=0.481). Conclusion: The Brody's scoring system for high resolution computed tomography seems to be a sensitive and efficient method to evaluate the progression of CF, and can be more reliable when we combine the CT scores with clinical parameter

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

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

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

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