Sensitivity to Oxidative Stress in DJ-1-Deficient Dopamine Neurons: An ES- Derived Cell Model of Primary Parkinsonism

1Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by rigidity, slowed movement, gait difficulty, and tremor at rest. The pathological hallmark of PD is the relatively selective loss of dopamine neurons (DNs) in the substantia nigra pars compacta in the ventral midbrain. Although the cause of neurodegeneration in PD is unknown, a Mendelian inheritance pattern is observed in approximately 5% of patients, suggesting a genetic factor. Extremely rare cases of PD have been associated with the toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which is taken up specifically by DNs through the dopamine transporter and is thought to induce cellular oxidative stress. Population-based epidemiological studies have further supported roles for genetic and environmental mechanisms in the etiology of PD.

2The identification of several genes that underlie familial forms of PD has allowed for the molecular dissection of mechanisms of DN survival. Autosomal dominant mutations in α-synuclein lead to a rare familial form of PD, and there is evidence that these mutations generate toxic, abnormal protein aggregates and cause proteasomal dysfunction. A majority of patients with sporadic PD harbor prominent intracytoplasmic inclusions, termed Lewy bodies, enriched for α-synuclein, as well as neurofilament protein. Mutations in a second gene, Parkin, lead to autosomal recessive PD. Parkin is a ubiquitin ligase that appears to participate in the proteasome-mediated degradation of several substrates.

3Homozygous mutations in a third gene, DJ-1, were recently associated with autosomal recessive primary parkinsonism. DJ-1 encodes a ThiJ domain protein of 189 amino acids that is broadly expressed in mammalian tissues. Interestingly, DJ-1 was independently identified in a screen for human endothelial cell proteins that are modified with respect to isoelectric point in response to sublethal doses of paraquat, a toxin that generates reactive oxygen species (ROS) within cells and has been associated with DN toxicity. Gene expression of a yeast homolog of DJ-1, YDR533C, is upregulated in response to sorbic acid, an inducer of cellular oxidative stress. These results suggest a causal role for DJ-1 in the cellular oxidative stress response.

4Surprisingly, animal models that harbor genetic lesions that mimic inherited forms of human PD, such as homozygous deletions in Parkin or overexpression of α-synuclein, have failed to recapitulate the loss of dopamine cells. An alternative approach, the genetic modification of midbrain DNs in vitro, is potentially useful but limited by the difficulty and variability in culturing primary postmitotic midbrain neurons. Other studies have focused on immortalized tumor cell lines, such as neuroblastoma cells, but these may not accurately model the survival of postmitotic midbrain neurons.

5Here we show that DJ-1-deficient cells display increased sensitivity to oxidative stress. DNs appear to be particularly sensitive to the loss of DJ-1. The initial accumulation of ROS is normal in DJ-1-deficient cells, but subsequent cellular defenses to ROS are impaired, leading to increased apoptosis.

6To investigate the normal cellular function of DJ-1 and the pathogenic mechanism of the PD mutations, we generated cells deficient in DJ-1. A murine embryonic stem (ES) cell clone, F063A04, that harbors a retroviral integration at the DJ-1 locus was obtained through the German Gene Trap Consortium (http://tikus.gsf.de) (Figure 1A; ). This integration is predicted to disrupt the normal splicing of DJ-1, leading to the generation of a truncated protein that lacks the carboxy-terminal domain required for dimerization and stability (unpublished data). Of note, a mutation that encodes a similarly truncated protein (at the human DJ-1 exon 7 splice acceptor) has been described in a patient with early-onset PD.

7To generate ES cell subclones homozygous for the trapped DJ-1 allele, clone F063A04 was exposed to a high dose of the antibiotic G418, which selects cells that are homozygous for the neomycin resistance gene insertion. Several homozygous mutant ES cell subclones (that had undergone gene conversion at the DJ-1 locus) were identified by Southern blotting (Figure 1B). To confirm that the trapped allele leads to the loss of wild-type (WT) DJ-1 protein, cell lysates from homozygous DJ-1-deficient (also termed “knockout” here) ES cell clones as well as the parental heterozygous clone were analyzed by Western blotting using polyclonal antibodies to the amino-terminal region of DJ-1 (amino acids 64–82) or full-length DJ-1 protein (unpublished data). Neither full-length nor truncated DJ-1 protein products were detected in knockout clones (Figure 1C), consistent with instability of the predicted truncated DJ-1 product. In addition, no full-length DJ-1 RNA was detected in cultures of knockout cells (Figure S1). In contrast, heterozygous and WT ES cells express high levels of DJ-1. Initial phenotypic analysis of knockout subclones indicated that DJ-1 is not essential to the growth rate of ES cells in culture, consistent with the viability of humans homozygous for DJ-1 mutations.

8DJ-1 has been hypothesized to function in the cellular response to oxidative stress. To investigate the role of DJ-1 in the oxidative stress response in vivo, DJ-1-deficient knockout and heterozygous ES cell clones were analyzed for cell viability in the context of increasing concentrations of H2O2. Heterozygous cells were used as controls because the knockout subclones were derived from these. Cell viability was initially determined by MTT assay (which detects reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT] by metabolic enzymes) in triplicate. Exposure to H2O2 led to significantly greater toxicity in the DJ-1-deficient cells; similar results were obtained with multiple knockout subclones in independent experiments (Figures 1D and 2A). In contrast, in the absence of toxin, heterozygous and knockout cells displayed comparable viability in the MTT assay (Figure S2). Consistent with the MTT assay, fluorescence-activated cell sorting (FACS) analysis of cells stained with annexin V (AV) and propidium iodide (PI) revealed increased death of knockout cells compared to heterozygous cells in the context of H2O2 exposure (Figure 1E). The increase in AV-positive cells implicated an apoptotic mechanism of cell death (Figure 1F). Furthermore, when exposed to H2O2, knockout cells displayed potentiated cleavage of poly(ADP-ribose)polymerase-1 (PARP) in a pattern indicative of an apoptotic death program (Figure 1G).

9Additional toxin exposure studies demonstrated that DJ-1-deficient cells were sensitized to the proteasomal inhibitor lactacystin (Figure 2B), as well as to copper (Figure S2), which catalyzes the production of ROS. We did not observe altered sensitivity to several other toxins, including tunicamycin (an inducer of the unfolded protein response in the endoplasmic reticulum; Figure 2C), staurosporine (a general kinase inhibitor that induces apoptosis) (Figure S2), or cycloheximide (an inhibitor of protein translation) (unpublished data).

10To confirm that altered sensitivity to oxidative stress is a consequence of the loss of DJ-1, we performed rescue experiments by overexpressing WT or mutant human DJ-1 in knockout ES cells. Plasmids encoding human Flag-tagged WT DJ-1, Flag-tagged PD-associated L166P mutant DJ-1, or vector alone, were transiently transfected into DJ-1-deficient clones, and these were subsequently assayed for sensitivity to H2O2 using the MTT viability assay. DJ-1-deficient cells transfected with a vector encoding Flag-WT human DJ-1 were effectively rescued in terms of viability in the presence of H2O2 (Figure 2D); Thus, viability in rescued knockout cells mimicked the viability of untransfected heterozygous cells in the context of H2O2 treatment (Figure 2A and 2D). In contrast, transfection of knockout cells with a vector encoding the PD-associated L166P mutant DJ-1 did not significantly increase the viability of H2O2-treated knockout cells (Figure 2D). Baseline cell viability in the absence of toxin exposure was not altered by DJ-1 overexpression, and Western blotting of lysates from transfected cells with an antibody specific to human DJ-1 demonstrated that transfected Flag-WT DJ-1 and Flag-L166P mutant DJ-1 accumulated comparably (Figure S2).

11We hypothesized that DJ-1 either alters the initial accumulation of intracellular ROS in response to H2O2 exposure, or that it functions downstream of the ROS burst and protects cells from consequent damage. Therefore, we quantified the accumulation of ROS in response to H2O2 treatment in knockout and heterozygous cells using the ROS-sensitive fluorescent indicator dye dihydrorhodamine-123 (DHR) and FACS analysis. Initial ROS accumulation (at 15 min after stimulation) appeared unaltered in the DJ-1-deficient cells in comparison to control heterozygous cells (Figure 2E). Consistent with this, accumulation of protein carbonyls, an index of oxidative damage to proteins, appeared normal initially (at 1 h after toxin exposure; Figure 2F). However, at 6 h after toxin exposure, a point at which knockout cells already display increased apoptosis (as indicated by PARP cleavage; see Figure 1G), protein carbonyl accumulation was robustly increased in the DJ-1-deficient cells. These data suggest that initial ROS accumulation is not altered by DJ-1 deficiency, but that the mutant cells are unable to appropriately cope with the consequent damage. Consistent with this result, no antioxidant or peroxiredoxin activity with purified DJ-1 protein in vitro was detected (S.S. and A.A., personal communication).

12Several methods have been established for the differentiation of ES cells into DNs in vitro. To extend our analysis of DJ-1 function to DNs, we differentiated DJ-1-deficient ES cells or control heterozygous cells into DNs in vitro by coculture with stromal cell–derived inducing activity (SDIA; Figure 3A). DNs were quantified by immunohistochemistry for tyrosine hydroxylase (TH; a marker for DNs and other catecholaminergic cells), or by analysis of dopamine transporter uptake activity (a quantitative DN marker). Production of DNs appeared to be significantly reduced in knockout ES cell cultures compared to parental heterozygous cultures at 18 days in vitro (DIV) as determined by both dopamine uptake and TH immunoreactivity (Figures 3B and 3C; 4A–4L). In contrast, general neuronal production did not appear altered in this assay in terms of the postmitotic neuronal marker TuJ1 (a monoclonal antibody specific to neuronal, not glial, class III β-tubulin) (Figures 3E and 4A–4L′); other neuronal subtypes also appeared normal, including GABAergic (Figures 3D and 4A′–4L′) and motor neurons (HB9-positive; Figure S3). To investigate whether the reduction in DNs in DJ-1-deficient cultures is due to defective generation or survival, a time course analysis was performed. At early time points (8 and 12 DIV), dopamine uptake activity was comparable in WT and DJ-1-deficient cultures, whereas subsequently the DJ-1-deficient cultures appeared defective (Figure 3F). Consistent with this, intracellular dopamine accumulation (as quantified using high-performance liquid chromatography) was significantly reduced in DJ-1-deficient cultures (6.4 ± 1.5 ng dopamine/mg protein) relative to control heterozygous cultures (66.0 ± 17.4 ng/mg) at 35 DIV. These data strongly suggest that DJ-1 deficiency leads to loss of DNs, rather than simply to downregulation of cell marker expression.

13We hypothesized that DJ-1-deficient DNs may be sensitized to oxidative stress, akin to DJ-1-deficient undifferentiated ES cells. To test this, DN cultures from DJ-1-deficient or heterozygous control ES cell cultures at 9 DIV were exposed to oxidative stress in the form of 6-hydroxydopamine (6-OHDA), a DN-specific toxin that enters DNs through the dopamine transporter and leads to oxidative stress and apoptotic death. DJ-1-deficient DNs displayed an increased sensitivity to oxidative stress in this assay (Figure 3G). Post-hoc analysis of the data indicates that the difference among genotypes is maximal at an intermediate dose of toxin (50 μM); at the highest dose of 6-OHDA employed (100 μM), the difference is lessened (because the heterozygote is increasingly affected as well), indicating that DJ-1-mediated protection is limited. Although we cannot exclude a role for DJ-1 in the late-stage differentiation of DNs, these data suggest that DJ-1 deficiency leads to reduced DN survival and predisposes these cells to endogenous and exogenous toxic insults.

14To confirm the role of DJ-1 in primary midbrain DNs, DJ-1 expression was inhibited by RNA interference (RNAi) in embryonic day 13.5 (E13.5) murine primary midbrain cultures by lentiviral transduction of short hairpin RNAs (shRNAs) (Figure 5). E13.5 midbrain cultures were transduced with a lentiviral vector that includes a gene encoding the green fluorescent protein marker eGFP, along with shRNAs homologous to murine DJ-1. DJ1-shRNA virus-infected cells displayed efficient silencing of DJ-1 gene expression to 10%–20% of control vector-infected cultures (as determined by Western blotting [Figure 5Q]). Transduction efficiency, as assessed by visualization of the fluorescent eGFP marker, exceeded 95% in all cases (Figure 5I and unpublished data). After 7 DIV, cultures were exposed to H2O2 for 24 h and then evaluated for DN survival as quantified by immunostaining for TH and dopamine transporter (DAT).

15Midbrain cultures transduced with DJ-1 shRNA virus and with control vector displayed similar numbers of TH-positive neurons in the absence of exposure to H2O2 (Figure 5A–D, 5I–L, and 5R–S). In contrast, in the presence of H2O2, DJ-1-deficient cultures displayed significantly reduced DN survival as quantified by immunohistochemistry for TH or DAT (Figure 5E–H, 5M–P, 5R–S). These studies were repeated three times with similar results. The reduction in DAT immunoreactivity appears to be more robust than the reduction in TH-positive cell number in the context of H2O2; this may reflect the differential localization of DAT to DN processes, whereas TH is primarily in the cell body. As we described in a previous manuscript, nondopaminergic cells in the E13.5 primary midbrain cultures are predominantly GABAergic neurons (90%–95%). Total embryonic midbrain neurons transduced with either DJ-1 shRNA or vector displayed comparable survival in the context of toxin exposure, suggesting that DJ-1 deficiency leads to a relatively specific alteration in DN survival (Figure 5T). These data are consistent with the analyses of ES cell–derived DNs above and indicate that DJ-1 is required for the normal survival of midbrain DNs in the context of toxin exposure.

16In this study we present evidence that DJ-1 is an essential component of the oxidative stress response of DNs. DJ-1-deficient cells display an apparently normal initial burst of ROS in response to H2O2, but they are unable to cope with the consequent toxicity, culminating in apoptosis. Additionally, we find that DJ-1 deficiency sensitizes cells to the proteasomal inhibitor lactacystin but not other toxic stimuli such as tunicamycin. Proteasomal inhibition induces the accumulation of short-lived and misfolded cytoplasmic proteins, leading to oxidative stress and apoptosis. ROS and proteasomal inhibition have previously been correlated with PD pathology, and it is therefore tempting to hypothesize that DJ-1 mutations lead to PD because of an increased sensitivity to such stressors.

17The apparent cell-type specificity of DN impairment in patients with the Parkinsonism-associated DJ-1 mutation is not predicted by the ubiquitous expression of DJ-1. In this study, we find that DJ-1 protects both dopaminergic and nondopaminergic cells from oxidative insult. However, DJ-1-deficient DNs appear to be especially sensitive to oxidative insult, suggesting relative cell-type specificity to the consequences of DJ-1 deficiency. Similar results are observed in DJ-1-knockout ES cell–derived DNs (which are devoid of any detectable DJ-1) and in primary DNs with DJ-1 levels reduced by RNAi “knockdown.” However, we find that even in the absence of exogenous toxin exposure, the knockout ES cell–derived DNs display reduced survival, whereas survival of the primary embryonic midbrain RNAi knockdown DNs appears to be similar to WT cells. We hypothesize that this discrepancy reflects the activity of residual DJ-1 (approximately 10%–20%) in the RNAi knockdown cultures. Alternatively, the knockout ES cell–derived DNs may be exposed to a greater degree of oxidative stress in vitro than are the knockdown-derived DNs even in the absence of added toxin. The mechanism by which DNs are preferentially targeted for destruction in the absence of DJ-1 is unclear. It has been proposed that DNs are subject to high levels of endogenous oxidative stress that may relate to dopamine metabolism.

18DJ-1 is structurally modified in the context of cellular oxidative stress, suggesting a possible function. Two recent studies investigated the role of DJ-1 in the oxidative stress response of neuroblastoma tumor cells. Both studies used RNAi to perturb the expression of DJ-1 in neuroblastoma tumor cell lines, and suggested that DJ-1 deficiency sensitizes cells to oxidative stress; these results are consistent with our data. further reported that overexpression of DJ-1 in neuroblastoma cells leads to a reduction in ROS accumulation and hypothesized that DJ-1 may harbor antioxidant activity in vivo. In contrast, we find that ES cells that are deficient in DJ-1 display a normal initial burst of ROS in the context of H2O2. Consistent with this, we fail to detect DJ-1 antioxidant activity in vitro.

19Finally, this study presents a novel, ES cell-based genetic approach to the study of neurodegenerative disorders. Mouse genetic models of disease are often limited by the inherent variability of animal experiments, the limited mouse life span, and the difficulties in manipulating whole animals. For instance, genetic rescue experiments and toxicological dose-response studies are impractical in whole animals. Furthermore, genetic cell models are more readily amenable to molecular dissection of disease mechanisms than are whole animals. Thus, genetically altered, ES cell–derived neurons are likely to be generally useful as cellular models of neurodegenerative disorders. Future studies may also utilize available human ES cells to investigate species differences.

20Undifferentiated ES cells were cultured using standard techniques. SDIA differentiation of ES cell cultures to DNs was performed as described in, except that ES cells were plated at a density of 500 cells/cm2 rather than approximatively 1,000 cells/cm2, and were cocultured with the MS5 mouse stromal cell line. For rescue experiments, cells were plated at a cell density of 1.4 × 106 cells/well. Transfections with plasmids encoding human Flag-WT DJ-1, PD-associated L166P mutant DJ-1, or vector alone, were performed using Lipofectamine 2000 (Life Technologies) for 18–36 h according to the manufacturer's instructions. 24 hours post-transfection, cells were split into 96-well plates and treated as described below. Primary cultures and lentiviral transductions were performed as described in.

21The pT1ATGβgeo gene trap vector, which includes βgeo, a fusion of the genes for β-galactosidase and neomycin resistance, is present between exons 6 and 7 of the murine DJ-1 gene, as determined by cDNA sequencing of trapped transcripts and genomic analysis (Figure 1A). To select for ES cell subclones homozygous for the trapped DJ-1 allele, we treated clone F063A04 with 4 mg/ml G418. Several subclones that were homozygous for the mutant DJ-1 allele were identified by Southern blotting (see Figure 1B), and three were chosen for further experimentation: clones 9, 23, and 32. To confirm that the trapped allele leads to the loss of wild-type (WT) DJ-1 protein, cell lysates from these clones, as well as from the parental heterozygous clone, were analyzed by Western blotting using polyclonal antibodies to the amino-terminal region of DJ-1 or the full-length DJ-1 protein. For Western blotting, cells were resuspended in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.2% Triton X-100, and incubated at 4 °C, rotating for 20 min. Cleared lysate was prepared by centrifuging the lysate at 13,000 rpm for 10 min at 4 °C.

22A rabbit polyclonal antibody to DJ-1 was generated against the synthetic polypeptide QNLSESPMVKEILKEQESR, which corresponds to amino acids 64–82 of the mouse DJ-1 protein. Antiserum was produced by the Polyquick polyclonal antibody production service of Zymed Laboratories (South San Francisco, California, United States). The antiserum was affinity-purified on a peptide-coupled Sulfolink column (Pierce Biotechnology, Rockford, Illinois, United States) according to the manufacturer's instructions. Antibody was used at a dilution of 1:200 for immunohistochemistry and Western blotting as described. Immunohistochemistry was performed with a rabbit polyclonal antibody to TH (PelFreez, Rogers, Arizona, United States; dilution 1:1000), the mouse monoclonal antibody to neuronal class III β-tubulin TuJ1 (Covance, Princeton, New Jersey, United States; dilution 1:500), and a rabbit polyclonal antibody to GABA (Sigma, St. Louis, Missouri, United States; dilution 1:1000). Western blotting was performed using a polyclonal antibody to cleaved PARP (Cell Signaling Technology, Beverly, Massachusetts, United States; dilution 1:500), a monoclonal antibody to DJ-1 (Stressgen Biotechnologies, San Diego, California, United States; dilution 1:1000), and a mouse monoclonal antibody to β-actin (Sigma, 1:500).

23ES cells plated in 96-well format (2.3 × 104 cells/well) were treated for 15 h with H2O2 in ES cell medium deficient in β-mercaptoethanol. Cell viability (as a percent of untreated control) was determined by MTT assay in triplicate. AV/PI (Molecular Probes, Eugene, Oregon, United States) staining was performed according to the manufacturer's instructions. For DHR staining (Molecular Probes), cells were preincubated for 30 min with DHR (5 μM), washed with PBS, then treated with H2O2 in ES cell medium deficient in β-mercaptoethanol for 15 min at 37 °C. The FACS analysis was performed using a FACSTAR sorter (Becton-Dickinson, Palo Alto, California, United States). Dopamine uptake assays were performed as described. Reported values represent specific uptake from which nonspecific uptake, determined in the presence of mazindol, was subtracted. Uptake values were normalized for protein content with the BCA kit (Pierce).

24For 6- hydroxydopamine (6-OHDA, Sigma) treatment, the drug was diluted in the differentiation medium and medium was changed every day for 72 h.

25Primary midbrain embryonic cultures were prepared and transduced with lentiviral vectors as described in. The DJ-1 shRNA vector was generated by insertion of annealed oligonucleotides 5′-TGTCACTGTTGCAGGCTTGGTTCAAGAGACCAAGCCTGCAACAGTGACTTTTTTC-3′ and 5′-ACAGTGACAACGTCCGAACCAAGTTCTCTGGTTCGGACGTTGTCACTGAAAAAAGAGCT-3′ into the LentiLox vector. For cellular dopamine quantification, cultures were incubated in standard differentiation medium supplemented with L-DOPA (50 μM) for 1 h to amplify dopamine production, as described in. Subsequently, cells were washed in PBS and then lysed in 0.2 M perchloric acid. Dopamine levels were quantified by HPLC and normalized for protein content as above.

26The cDNA for human DJ-1 was PCR-amplified from a human liver cDNA library (Clontech, Palo Alto, California, United States). For expression of DJ-1 in ES cell rescue experiments, DJ-1 was cloned into the expression vector pcDNA3.1 (Invitrogen, Carlsbad, California, United States) containing a Flag peptide sequence at the N-terminus using standard cloning techniques. Flag-L166P DJ-1 (pcDNA3) was generated by PCR-mediated mutagenesis.

27For protein carbonyl quantitation, cells were plated (1.4 × 105 cells per well), grown for 24 h, and then treated with 10 μM H2O2 as indicated. Cells were lysed in 200 μl lysis buffer and cleared lysate was prepared as described above. An aliquot of 40 μl from each time point was added to 2 M HCl (120 μl) with or without 10 μM 2,4-dinitrophenyl-hydrazine and incubated for 1 h at 24 °C with shaking. Proteins were then TCA-precipitated and resuspended in 200 μl of 6 M guanidinium chloride. Absorbance was measured at 360 nm, and DNP-conjugated samples were normalized for protein concentration with the underivitized control samples.

28The GenBank (http://www.ncbi.nlm.nih.gov/) accession numbers of the genes discussed in this paper are α-synuclein (NM_000345), Parkin (AB009973), and DJ-1 (AB073864).