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

AMP-Activated Protein Kinase-Regulated Activation of the PGC-1α Promoter in Skeletal Muscle Cells

The mechanisms by which PGC-1α gene expression is controlled in skeletal muscle remains largely undefined. Thus, we sought to investigate the transcriptional regulation of PGC-1α using AICAR, an activator of AMPK, that is known to increase PGC-1α expression. A 2.2 kb fragment of the human PGC-1α promoter was cloned and sequence analysis revealed that this TATA-less sequence houses putative consensus sites including a GC-box, a CRE, several IRSs, a SRE, binding sites for GATA, MEF2, p 53, NF-κB, and EBox binding proteins. AMPK activation for 24 hours increased PGC-1α promoter activity with concomitant increases in mRNA expression. The effect of AICAR on transcriptional activation was mediated by an overlapping GATA/EBox binding site at −495 within the PGC-1α promoter based on gel shift analyses that revealed increases in GATA/EBox DNA binding. Mutation of the EBox within the GATA/EBox binding site in the promoter reduced basal promoter activity and completely abolished the AICAR effect. Supershift analyses identified USF-1 as a DNA binding transcription factor potentially involved in regulating PGC-1α promoter activity, which was confirmed in vivo by ChIP. Overexpression of either GATA-4 or USF-1 alone increased the p851 PGC-1α promoter activity by 1.7- and 2.0-fold respectively, while co-expression of GATA-4 and USF-1 led to an additive increase in PGC-1α promoter activity. The USF-1-mediated increase in PGC-1α promoter activation led to similar increases at the mRNA level. Our data identify a novel AMPK-mediated regulatory pathway that regulates PGC-1α gene expression. This could represent a potential therapeutic target to control PGC-1α expression in skeletal muscle

Effect of GATA-4 and USF-1 overexpression on PGC-1α promoter activity and mRNA expression.

<p>A. Representative western blots of protein extracts made from C₂C₁₂ cells transfected with either 2 or 4 µg of GATA-4 or USF-1 or an empty vector (EV) control. B. USF-1 and GATA-4 were co-transfected with the pGL3 (EV; 500ng) or the p851 PGC-1α promoter reporter construct (500ng) along with the appropriate empty vector controls. Relative luciferase activities were measured 48 hours after transfection and are plotted as the fold change above empty vector. Values are means±SEM, (n = 8); * p<0.05 versus p851-EV and #, p<0.05 versus p851-USF-1 or p851-GATA-4. C. Cells were transfected with 4 µg of USF-1 or an empty vector (EV) control. EtBr-stained DNA gel of PGC-1α amplified by PCR from EV- and USF-1 transfected cells. s12rRNA was used to verify equal loading. Data are representative of one experiment with conditions repeated in duplicate (AU = arbitrary units).</p

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