DJ-1 In Vitro Chaperone Activity and In Vivo Oxidative Stress Protection Activity Require Cysteine 53 but Not Cysteine 106

<div><p>(A) DJ-1 cysteine-to-alanine mutants C106A, C53A, and a triple mutant that harbors mutations at all three cysteines in DJ-1 (C106A/C53A/C46A), as well as L166P, were tested for in vitro chaperone activity by CS aggregation suppression assay.</p> <p>(B) Self-association of DJ-1 cysteine mutants. Murine neuroblastoma CAD cells were transiently cotransfected with Flag-tagged human DJ-1 vectors (either WT or mutant) along with WT YFP-tagged human DJ-1. Lysates were immunoprecipitated with anti-Flag antibodies and probed by Western blotting with an antibody specific for human DJ-1. WT Flag-DJ-1, C106A DJ-1, C53A DJ-1, and C106A/C53A/C46A DJ-1 effectively coprecipitated WT GFP-DJ-1, whereas the L166P mutant Flag-DJ-1 failed to do so. Lysates represent 20% of the input for the immunoprecipitate; Flag-DJ-1 migrates at 22 kDa, and YFP-DJ-1 migrates at 50 kDa.</p> <p>(C) DJ-1-deficient ES cells were transiently transfected with vector alone, WT DJ-1, or DJ-1 cysteine mutants, and exposed to 10 μM H₂O₂ for 15 h followed by MTT assay. The viability of the cells in the absence of drug treatment was not altered by the expression of WT or mutant DJ-1). Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test. * <i>p</i> < 0.05</p> <p>(D) Expression levels of WT and mutant forms of DJ-1 were comparable as determined by Western blotting for human DJ-1 and β-actin.</p></div

DJ-1 Inhibits Formation of αSyn Intracytoplasmic Inclusions

<div><p>(A–L) CAD murine neuroblastoma cells were transfected with WT DJ-1 (A–F), L166P DJ-1 (G–I) or vector control (J–L), along with Flag-αSyn (D–L) or vector control (A–C) and differentiated in vitro by serum withdrawal for 72 h. Cells were fixed and stained with a mouse monoclonal antibody for αSyn and ToPro3, a nuclear dye, and images were obtained by confocal microscopy. Transfection of Flag-αSyn induced formation of intracytoplasmic inclusions (arrows). Scale bar, 20 μm.</p> <p>(M) Quantification of cells with inclusions was performed on ten random images from each of three wells per condition. Images were quantified by an observer blinded to the experiment. A significantly lower percentage of cells harbor inclusions in the context of WT DJ-1 overexpression. Aggregation is expressed as the percentage of cells containing αSyn aggregates per frame. Total cell number per frame, as determined by ToPro3 staining, did not differ significantly (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020362#sg003" target="_blank">Figure S3</a>). Data are shown as the mean ± SEM, and were analyzed by ANOVA with Fisher's post-hoc test. * <i>p</i> < 0.05.</p> <p>(N–S) Cells were fixed and stained with a monoclonal antibody for αSyn and a polyclonal antibody that recognizes both transfected human DJ-1 and endogenous murine DJ-1. DJ-1 does not appear to colocalize with the αSyn aggregates. Scale bar, 20 μm.</p></div

DJ-1 Inhibits Formation of NFL Intracytoplasmic Inclusions

<div><p>(A–L) CAD cells were transfected with an aggregation-prone mutant NFL (Q333P) plasmid, as well as WT human DJ-1 plasmid (that also harbors GFP; E–H), L166P mutant DJ-1 (that also harbors GFP; I–L), or control GFP vector (A–D). After 72 h in culture, cells were fixed and stained with a mouse monoclonal antibody for NFL and ToPro3, a nuclear dye. Scale bar, 100 μm.</p> <p>(M–R) CAD cell transfectants, as above, were fixed and stained with a polyclonal antibody for NFL (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020362#pbio-0020362-Perez-Olle1" target="_blank">Perez-Olle et al. 2002</a>) along with a mouse monoclonal antibody specific for the transfected human DJ-1. Scale bar, 20 μm.</p> <p>(S) Quantification of CAD cell NFL aggregates was performed using confocal microscopy. Images from tenrandomly selected fields in each of three wells were quantified for the presence of aggregates for each condition and presented as a percentage of total cells per field. Total cell number was determined by ToPro3 nuclear staining and did not differ significantly (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020362#sg003" target="_blank">Figure S3</a>). Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test. * <i>p</i> < 0.05.</p></div

DJ-1 Inhibits Formation of αSyn Protofibrils and Fibrils In Vitro

<div><p>(A) Purified αSyn (200 μM) was incubated for 2 h at 55 °C in the presence of WT DJ-1, L166P mutant DJ-1, GST, or Hsp27 (all at 100 μM). WT DJ-1 inhibits accumulation of αSyn protofibrils in vitro, while L166P mutant DJ-1, GST, and Hsp27 do not.</p> <p>(B) Suppression of αSyn protofibril formation by WT DJ-1 (in triplicate) was quantified as compared to GST (as a negative control) and mutant L166P DJ-1. Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test. * <i>p</i> < 0.05.</p> <p>(C) Purified αSyn (200 μM) was incubated for 1 wk at 37 °C in the presence of WT DJ-1, L166P mutant DJ-1, or GST (all at 100 μM). WT DJ-1 inhibits formation of mature Congo red–positive αSyn fibrils. Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test. * <i>p</i> < 0.05.</p></div

Overexpression of WT DJ-1 Inhibits Aggregation of αSyn In Vivo

<div><p>(A) CAD murine neuroblastoma cells were transfected with Flag-αSyn along with WT DJ-1, L166P clinical mutant, or vector alone, and were differentiated in vitro via serum withdrawal. Cells were subsequently treated with 2 mM FeCl₂ (Fe), 5 μM lactacystin (LC), or media alone (0). Triton X-100-soluble (Tx-100 sol) and Triton X-100-insoluble (Tx-100 insol) fractions were analyzed by Western blotting. Upon FeCl₂ treatment, αSyn accumulates in the Triton X-100-insoluble fraction, and accumulation of insoluble αSyn is inhibited by overexpression of WT DJ-1 (left) but not the L166P clinical mutant (right).</p> <p>(B) Triton X-100-insoluble αSyn as quantified by NIH Image J of a Western blot (from [A]).</p> <p>(C) Heterozygous (+/–) and DJ-1 deficient (–/–) ES cells were differentiated using the embryoid body protocol. Cells were transfected with Flag-αSyn (F-αSyn), and, after 48 h, treated with 2 mM FeCl₂ or with media alone for 18 h. Cell lysates were analyzed by Western blotting for αSyn or β-actin. In the Triton X-100-soluble fraction (Tx-100 sol), DJ-1 accumulated to a similar extent in the knockout and control cells. In contrast, αSyn accumulation in the insoluble pool (Tx-100 insol) was detectable only in the knockout cells, and this was further promoted by FeCl₂ treatment.</p> <p>(D) CAD cells transfected with Flag-αSyn (F-αSyn) along with WT DJ-1 (or vector alone) were treated with 2 mM FeCl₂ or media alone for 18 h. Triton X-100-soluble cell lysates were immunoprecipitated with a mouse monoclonal antibody for the Flag epitope and Western blotted for DJ-1. FeCl₂ treatment induces association of Flag-αSyn with WT DJ-1. Lysates represent 20% input of the immunoprecipitation (IP α-Flag). The Triton X-100 soluble pool of DJ-1 is reduced by αSyn overexpression (but not vector control), particularly in the context of FeCl₂ treatment (bottom).</p> <p>(E) DJ-1 colocalizes with αSyn in the Triton X-100-insoluble fraction upon FeCl₂ treatment. The Western blot from (A) was stripped and reprobed for DJ-1.</p></div

DJ-1 Is a Redox-Dependent Molecular Chaperone That Inhibits α-Synuclein Aggregate Formation

<div><p>Parkinson's disease (PD) pathology is characterized by the degeneration of midbrain dopamine neurons (DNs) ultimately leading to a progressive movement disorder in patients. The etiology of DN loss in sporadic PD is unknown, although it is hypothesized that aberrant protein aggregation and cellular oxidative stress may promote DN degeneration. Homozygous mutations in <em>DJ-1</em> were recently described in two families with autosomal recessive inherited PD (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020362#pbio-0020362-Bonifati1">Bonifati et al. 2003</a>). In a companion article (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020362#pbio-0020362-Martinat1">Martinat et al. 2004</a>), we show that mutations in DJ-1 alter the cellular response to oxidative stress and proteasomal inhibition. Here we show that DJ-1 functions as a redox-sensitive molecular chaperone that is activated in an oxidative cytoplasmic environment. We further demonstrate that DJ-1 chaperone activity in vivo extends to α-synuclein, a protein implicated in PD pathogenesis.</p> </div

Neuronal Differentiation of DJ-1-Deficient and Control Heterozygous ES Cell Cultures

<div><p>(A–L) <i>DJ-1</i> heterozygous (+/–; A–F) and knockout (–/– [clone 32]; G–L) cultures were differentiated by SDIA for 18 DIV and immunostained with antibodies to TH (green) and TuJ1 (red). Images of both (Merge) are also shown.</p> <p>(A′–L′) Immunostaining of <i>DJ-1</i> heterozygous (+/–, A′–F′) and deficient (–/–, G′–L′) cultures with antibodies for GABA (green) and TuJ1 (red). Scale bar, 50 μm. Images of both (Merge) are also shown.</p></div

DJ-1-Deficient ES Cells Are Sensitized to Oxidative Stress

<div><p>(A) Schematic map of the murine <i>DJ-1</i> gene in clone F063A04. The retroviral insertion places the engrailed-2 (En2) intron, the splice acceptor (SA), and the β-galactosidase/neomycin resistance gene fusion (β-geo) between exons 6 and 7.</p> <p>(B) Southern blot analysis of KpnI-digested genomic DNA from <i>DJ-1</i> homozygous mutant (–/–), WT (+/+), and heterozygous (+/–)cells, probed with murine <i>DJ-1</i> cDNA. WT DNA shows a predicted 14-kb band (WT), whereas the mutant allele migrates as a 9-kb band (insertion).</p> <p>(C) Western blot (WB) of ES cell lysates from WT (+/+), <i>DJ-1</i> heterozygous (+/–), and mutant homozygous (–/–) clones with antibodies to murine DJ-1 (α-DJ-1) or β-actin (α-β-actin). DJ-1 migrates at 20 kDa, β-actin at 45 kDa.</p> <p>(D) ES cells were exposed to 0, 5, 10, and 20 μM H₂O₂ for 15 h and viability was assayed by MTT. Responses of <i>DJ-1</i> heterozygous cells (diamonds) and <i>DJ-1</i> knockout clones 9 (open circles), 16 (solid circles), 23 (squares), and 32 (triangles) are shown. ** <i>p</i> ≤ 0.01; *** <i>p</i> ≤ 0.0001.</p> <p>(E and F) Cell death of <i>DJ-1</i> heterozygous and DJ-1-deficient cells (clone 32) after exposure to H₂O₂ (10 μM) was quantified by staining with PI and an antibody to AV with subsequent FACS analysis. AV staining marks cells undergoing apoptosis, whereas PI staining indicates dead cells. * <i>p</i> ≤ 0.05.</p> <p>(G) <i>DJ-1</i> heterozygous (+/–) and knockout (clone 32; –/–) cells were assayed at 1, 6, and 24 h after treatment with 10 μM H₂O₂ by Western blotting for cleaved PARP (89 kDa), which indicates apoptosis. No band is seen for cleaved PARP or β-actin for the DJ-1-deficient cells at 24 h due to cell death. Data represent means ± standard error of the mean (SEM) and were analyzed by ANOVA with Fisher's post-hoc test.</p></div

RNAi “Knockdown” of <i>DJ-1</i> in Primary Embryonic Midbrain DNs Display Increased Sensitivity to Oxidative Stress

<div><p>(A–P) Primary midbrain cultures from E13.5 embryos were infected with lentiviral vectors encoding <i>DJ-1</i> shRNA (or vector alone) under the regulation of the control vector (A–H) or the U6 promoter (I–P). Cells were cultured for 1 wk after infection and then exposed to H₂O₂ (5 μM; E–H and M–P) for 24 h. Cultures were immunostained for TH (B, F, J, and N) or DAT (C, G, K, or O) and visualized by confocal microscopy. Images containing all stains are included (Merge; D, H, L, and P). Scale bar, 100 μm.</p> <p>(Q) Cell lysates prepared from midbrain primary cultures infected with <i>DJ-1</i> shRNA lentivirus (or control vector) were analyzed by Western blotting for murine DJ-1 or β-actin.</p> <p>(R–T) Quantification of TH, DAT, and GFP signal was performed on ten randomly selected fields in each of three wells for each condition. Red triangles, <i>DJ-1</i> shRNA treated; black circles, control vector. Data represent the means ± SEM and were analyzed by ANOVA followed by Fisher's post-hoc test. * <i>p</i> ≤ 0.05.</p></div