Parkinson Disease-associated DJ-1 Is Required for the Expression of the Glial Cell Line-derived Neurotrophic Factor Receptor RET in Human Neuroblastoma Cells

Rossana Foti,Silvia Zucchelli,Marta Biagioli,Paola Roncaglia,Sandra Vilotti,Raffaella Calligaris,Helena Krmac,Javier Enrique Girardini,Giannino Del Sal,Stefano Gustincich

doi:10.1074/jbc.m109.088294

Abstract

Mutations in PARK7/DJ-1 are associated with autosomal recessive, early onset Parkinson disease (PD). DJ-1 is an atypical peroxiredoxin-like peroxidase that may act as a redox-dependent chaperone and a regulator of transcription. Here we show that DJ-1 plays an essential role in the expression of rearranged during transfection (RET), a receptor for the glial cell line-derived neurotrophic factor, a neuroprotective molecule for dopaminergic neurons, the main target of degeneration in PD. The inducible loss of DJ-1 triggers the establishment of hypoxia and the production of reactive oxygen species that stabilize the hypoxia-inducible factor-1alpha (HIF-1a). HIF-1a expression is required for RET down-regulation. This study establishes for the first time a molecular link between the lack of functional DJ-1 and the glial cell line-derived neurotrophic factor signaling pathway that may explain the adult-onset loss of dopaminergic neurons. Furthermore, it suggests that hypoxia may play an important role in PD.

Highlights

Armenise-Harvard Foundation, and the Italian Institute of Technology. □S The on-line version of this article contains supplemental Table S1 and Figs
Generation of Inducible Stable Cell Lines Expressing RNA Interference against DJ-1—To mimic the loss-of-function effects as seen in Parkinson disease (PD) patients with DJ-1 mutations, we used siDJ-1 constructs to inhibit the synthesis of endogenous DJ-1 in the human SH-SY5Y neuroblastoma cell line
As a further support to HIF-1a protein stabilization in siDJ1#1 cells, we found that some established HIF-1a target genes, like vascular endothelial growth factor A (VEGFA) and adrenomedullin (ADM), were up-regulated by DJ-1 loss

Summary

EXPERIMENTAL PROCEDURES

Constructs—Expression vectors encoding for pcDNA3-FLAG and pcDNA3–2ϫFLAG-DJ-1 wild type and L166P were previously described [20]. pcDNA3-FLAG-DJ-1 C106A mutant was generated by site-directed mutagenesis. Two different oligonucleotide sequences were selected for the silencing of DJ-1 expression using small interfering RNA Target Finder software (Invitrogen). SH-SY5Y cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. For preparation of SH-SY5Y-inducible cell lines, first we generated the acceptor cells by transfection of pcDNA6-TetR and selection with Blasticidin (3 ␮g/ml) (InvivoGen). We generated SH-SY5Y-inducible stable cell lines by transfecting linearized pSuperior containing interference oligonucleotides against DJ-1 and selecting for positive clones in the presence of 300 ␮g/ml G418 (Invitrogen) and 3 ␮g/ml Blasticidin (InvivoGen). For HIF-1a silencing, cells were transfected with Oligofectamine (Invitrogen) according to the manufacturer’s instructions. Data were imported in the MultiExperiment Viewer (MeV) software [24], and statistical analysis was performed with the SAM (Significance Analysis of Microarrays) module [25] to detect significantly differentially expressed genes. Each group was compared individually with reference control group using Student’s t test (Microsoft Excel software)

RESULTS

Affymetrix gene chip analysis

Gene symbol

DISCUSSION