DJ-1 gene deletion reveals that DJ-1 is an atypical peroxiredoxin-like peroxidase

Abstract

Parkinson's disease (PD) is a common neurodegenerative movement disorder. Whereas the majority of PD cases are sporadic, rare genetic defects have been linked to this prevalent movement disorder. Mutations in DJ-1 are associated with autosomal recessive early-onset PD. The exact biochemical function of DJ-1 has remained elusive. Here we report the generation of DJ-1 knockout (KO) mice by targeted deletion of exon 2 and exon 3. There is no observable degeneration of the central dopaminergic pathways, and the mice are anatomically and behaviorally similar to WT mice. Fluorescent Amplex red measurements of H(2)O(2) indicate that isolated mitochondria from young and old DJ-1 KO mice have a 2-fold increase in H(2)O(2). DJ-1 KO mice of 2-3 months of age have a 60% reduction in mitochondrial aconitase activity without compromising other mitochondrial processes. At an early age there are no differences in antioxidant enzymes, but in older mice there is an up-regulation of mitochondrial manganese superoxide dismutase and glutathione peroxidase and a 2-fold increase in mitochondrial glutathione peroxidase activity. Mutational analysis and mass spectrometry reveal that DJ-1 is an atypical peroxiredoxin-like peroxidase that scavenges H(2)O(2) through oxidation of Cys-106. In vivo there is an increase of DJ-1 oxidized at Cys-106 after 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine intoxication of WT mice. Taken together these data indicate that the DJ-1 KO mice have a deficit in scavenging mitochondrial H(2)O(2) due to the physiological function of DJ-1 as an atypical peroxiredoxin-like peroxidase.

Fig. 1.

Increase in H₂O₂ production and deficits in aconitase activity. (A) Significant increase of mitochondrial H₂O₂ production in KO mice compared with WT littermates; brain-isolated mitochondria were incubated in the presence or absence of rotenone in young mice (2–3 months). (B) Brain mitochondria from KO and WT mice (2–3 months old) were incubated in the presence or absence of succinate or succinate plus malonate. (C) Comparison of the mitochondrial H₂O₂ production in young (2–3 months) and aged (18–24 months) mice. H₂O₂ production was measured by using Amplex red (Molecular Probes, Eugene, OR). H₂O₂ production was calculated from a standard curve generated from known concentrations of H₂O₂. (D) Mitochondrial aconitase activity in isolated mitochondria from brains of 2- to 3-month-old and 18- to 24-month-old WT and KO mice. Then mitochondrial aconitase was reactivated as described in Materials and Methods, and the activity was measured. (E) Mitochondrial proteins were subjected to immunoblot with densitometric analysis by using hsp70 as a loading control. No differences in protein expression were found. (F and G) Mitochondrial citrate synthase and fumarase activity of brains in 2- to 3-month-old and 18- to 24-month-old mice was measured. (H and I) No differences were found in glutamine synthase and α-ketoglutarate dehydrogenase activity between mitochondria isolated from 2- to 3-month-old KO mice and WT littermates. Significance was determined by a two-way ANOVA with the Student–Newman–Keuls test. *, P ≤ 0.05. Data are the means ± SEM (n = 3).

Fig. 2.

Compensatory mechanism in DJ-1 KO mice. Shown are immunoblot and densitometric analyses of the protein expression of GPx, MnSOD, and catalase in whole brain lysates with β-actin as a loading control (A) and VDAC as a loading control for the mitochondrial fraction (B) in aged mice (18–24 months old). Data are the means ± SEM (n = 3). Significance was determined by Student's t test. (C and D) Mitochondrial activity of antioxidant enzymes of young (2–3 months old) and aged (18–24 months old) WT and KO mice: GPx (C) and MnSOD (D). (E and F) Cytosolic levels of reduced (E) and oxidized (F) forms of glutathione in 2- to 3-month-old and 18- to 24-month-old mice. The enzyme activities are expressed in units per milligram of tissue whereas the reduced and oxidized forms of glutathione are expressed as nanograms per milligram of protein. Data represent the means ± SEM from samples of five different animals measured three times on different days. Significance was determined by ANOVA with the Student–Newman–Keuls test. *, P ≤ 0.05.

Fig. 3.

DJ-1 consumes H₂O₂. (A) Recombinant DJ-1 (5 μg/ml) incubated with 10 nM H₂O₂ reduced the H₂O₂ concentration in the reaction shown by the Amplex red assay. (B) Recombinant DJ-1 consumes H₂O₂ in a dose-dependent manner. This reaction is abolished by purified catalase. DJ-1 does not exhibit catalase-like activity as assessed by Clark electrode (data not show). (C) In the absence of HRP there is no signal, indicating that DJ-1 is not peroxidase-like. (D) Representative in-gel detection of cysteine residues labeled with Thioglo-1. Lane 1, Thioglo-1 without protein; lane 2, human DJ-1; lane 3, DJ-1 reacted with 250 μM hydrogen peroxide for 60 min at 37°C; lane 4, DJ-1 reacted with 250 and 500 μM t-butyl hydroperoxide for 60 min at 37°C; lane 5, DJ-1 reacted with 250 μM peroxynitrite for 5 min at 37°C. (E) Second-order rate constant determination for the reaction between 250 μM H₂O₂ and DJ-1 at 37°C using the times indicated. Catalase was added to remove excess H₂O₂, and samples were assayed for nonreactive cysteine by using Thioglo-1 labeling. Data represent the mean and SD from three different analyses. Significance was determined by ANOVA with the Student–Newman–Keuls test. *, P ≤ 0.05.