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Proc Natl Acad Sci U S A. Jul 5, 2005; 102(27): 9691–9696.
Published online Jun 27, 2005. doi:  10.1073/pnas.0409635102
PMCID: PMC1172235
Neuroscience

Interaction of DJ-1 with Daxx inhibits apoptosis signal-regulating kinase 1 activity and cell death

Abstract

Investigations into the cellular and molecular biology of genes that cause inherited forms of Parkinson's disease, as well as the downstream pathways that they trigger, shed considerable light on our understanding the fundamental determinants of life and death in dopaminergic neurons. Homozygous deletion or missense mutation in DJ-1 results in autosomal recessively inherited Parkinson's disease, suggesting that wild-type DJ-1 has a favorable role in maintaining these neurons. Here, we show that DJ-1 protects against oxidative stress-induced cell death, but that its relatively modest ability to quench reactive oxygen species is insufficient to account for its more robust cytoprotective effect. To elucidate the mechanism of this cell-preserving function, we have screened out the death protein Daxx as a DJ-1-interacting partner. We demonstrate that wild-type DJ-1 sequesters Daxx in the nucleus, prevents it from gaining access to the cytoplasm, from binding to and activating its effector kinase apoptosis signal-regulating kinase 1, and therefore, from triggering the ensuing death pathway. All these steps are impaired by the disease-causing L166P mutant isoform of DJ-1. These findings suggest that the regulated sequestration of Daxx in the nucleus and keeping apoptosis signal-regulating kinase 1 activation in check is a critical mechanism by which DJ-1 exerts its cytoprotective function.

Keywords: apoptosis, Parkinson's disease, neuroprotection, neurodegeneration, oxidative stress

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Although the majority of the cases appear to be sporadic, the disorder also can be associated with specific genetic defects, several of which have been identified, including α-synuclein, parkin, PINK1, dardarin/LRRK2, and DJ-1 (16).

Mutations in the DJ-1 gene were originally discovered in two pedigrees with inherited PD (6). In one, a large homozygous genomic deletion encompassing exons 1–5 of DJ-1 leads to the absence of the gene product; and in the other, a homozygous point mutation results in the substitution of a highly conserved leucine for a proline at position 166 (L166P). In both pedigrees, heterozygous individuals are unaffected, whereas all homozygotes have the disease, consistent with recessive inheritance. Thus, these mutations are expected to result in loss of DJ-1 function. Wild-type DJ-1 forms a homodimer, whereas the L166P mutation destabilizes the dimer interface (7), suggesting that dimerization is critical for the physiologic functions of this protein.

DJ-1, originally identified as an oncogene product, appears to be involved in diverse biological processes (8). First, several lines of evidence suggest that DJ-1 plays a role in the oxidative stress response (9, 10). In cultured mammalian cells, DJ-1 quenches reactive oxygen species and is converted into a variant with a more acidic isoelectric point (9, 11). Therefore, DJ-1 protects against reactive oxygen species-induced cell death, and its suppression with small interfering RNA (siRNA) sensitizes cells to such insults (1013). Second, DJ-1 modulates transcription through interaction with DJ-1-binding protein (14) as well as with protein inhibitor of activated STAT (PIAS) (15). The latter modulates the activity of various transcription factors. Third, DJ-1 has been recognized as a regulatory subunit of an RNA-binding protein (16). Fourth, DJ-1, which is structurally related to the molecular chaperone Hsp31, may have chaperone activity itself, preventing heat-induced aggregation of substrate proteins, including α-synuclein (17). Despite these various functions, the cell biologic phenomena and signaling pathways that are central for the neuroprotective effect of DJ-1 against oxidative stress are poorly understood. The present report endeavors to elucidate these mechanisms.

Materials and Methods

Plasmids. Full-length human DJ-1 cDNA was amplified by PCR from a human adult brain cDNA library (Invitrogen) by using primers 5′-GCCGAATTCCAAATGGCTTCCAAAAGAGCT-3′ and 5′-GGCGAATTCCTAGTCTTTAAGAACAAGTGG-3′ and cloned into pCMV-Tag2B (Stratagene), pEGFP-C2 (Clontech), and pEG202 to produce FLAG-tagged, GFP-tagged, or LexA-tagged DJ-1, respectively. Full-length human Daxx cDNA was cloned by PCR with IMAGE clone 2185666 (Invitrogen) as template by using Pfu DNA polymerase. Primers (5′-GGCGGTACCATGGCCACCGCTAACAGCATCATC-3′ and 5′-GCCGAATTCCTAATCAGAGTCTGAGAGCACGAT-3′) contained restriction enzyme cleavage sites (KpnI and EcoRI) to facilitate insertion of the product in pHM6 (Roche), expressing hemagglutinin (HA)-tagged Daxx.

Cell Viability/Death Assay. Viability of stably DJ-1-transfected cell lines was quantified by using the Cell Titer 96 Aqueous One solution cell proliferation assay kit (Promega). Cells were plated in 96-well tissue culture plates at a density of 5 × 104 cells per well and cultured for 24 h, followed by exposure to chemical insults for 24 h. After the cells were washed with PBS, Cell Titer 96 Aqueous One solution (Promega) was added, followed by incubation at 37°C. Absorbance was measured at 490 nm. Cell death assay for transiently DJ-1-transfected cells was done by using ethidium homodimer 1 (EthD-1) (Molecular Probes), which enters dead cells with damaged membranes and undergoes enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence. EthD-1 is excluded from living cells with intact plasma membranes. Cells transiently cotransfected with DJ-1 and EGFP plasmids were plated in four-well poly(d-lysine)-coated culture slides (Becton Dickinson) and cultured for 24 h, followed by H2O2 treatment for 24 h. After labeling with EthD-1, >200 EGFP-positive cells were counted. The percentage of cell death was calculated as the ratio of double-labeled EthD-1/EGFP-positive cells to EGFP-only positive cells.

siRNA Experiment. siRNA targeting DJ-1 (5′-UGGAGACGGUCAUCCCUGUdTdT-3′ and 5′-ACAGGGAUGACCGUCUCCAdTdT-3′) and mutated siRNA (5′-UGGAGACGGAGAUCCCUGUdTdT-3′ and 5′-ACAGGGAUCUCCGUCUCCAdTdT-3′) were synthesized (Invitrogen). Several concentrations of siRNAs were transiently transfected into SH-SY5Y cells by using Mirus TransIT-TKO reagent (Mirus, Madison, WI) according to the manufacturer's manual.

2′,7′-Dichlorofluorescein Diacetate (DCFH-DA) Assay. The intracellular level of H2O2 was measured by using DCFH-DA as described in ref. 18. Cells (5 × 104 per well) were plated into 96-well plates for a day, washed with KRH buffer, and then incubated with 100 μM DCFH-DA for 30 min. After removing DCFH-DA, cells were washed and incubated with different concentrations of H2O2 in KRH buffer for 1 h, and fluorescence (Ex485/Em535) was measured by using a Victor2 fluorometer (PerkinElmer).

Yeast Two-Hybrid Screen. Full-length human DJ-1 cDNA fused to the LexA DNA-binding domain in pEG202 was used as bait for two-hybrid screening. After confirming that the bait plasmid itself could not activate transcription from the GAL1–GAL10 promoter in the reporter plasmid pSH18-34, we screened a human adult brain cDNA library constructed in pYESTrp2 (Stratagene). β-Galactosidase activity was measured with lysed yeast cells by using the β-galactosidase assay kit (Promega) according to the manufacturer's protocol.

Apoptosis Signal-Regulating Kinase 1 (ASK1) Activity Assay. Cells transfected as indicated were lysed in a buffer containing 20 mM Tris·HCl (pH 7.5), 12 mM β-glycerophosphate, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 (Sigma), and a mixture of protease inhibitors and phosphatase inhibitors from Roche Molecular Biochemicals. Cell extracts were cleared by centrifugation at 20,000 × g for 15 min, and the supernatants were immunoprecipitated with either agarose-conjugated anti-Myc antibody (9E10) to pull down Myc-tagged ASK1 or with anti-ASK1 antibody (H-300) (Santa Cruz Biotechnology)/protein G-agarose to pull down endogenous ASK1. Immunoprecipitates were used in an in vitro kinase assay with a reaction buffer consisting of 20 mM Tris·HCl (pH 7.5) and 20 mM MgCl2 containing 0.5 μCi (1 Ci = 37 GBq) of [γ-32P]ATP for 20 min at 30°C by using myelin basic protein (40 μg/ml) as substrate. Samples were resolved in SDS/PAGE and subjected to autoradiography.

Immunoprecipitation and Western Blot Analysis. Transfected cells were lysed in a buffer containing PBS with 0.5% Nonidet P-40 (Sigma) and a mixture of protease inhibitors and phosphatase inhibitors from Roche Molecular Biochemicals. After homogenizing with 20 strokes by using a Dounce homogenizer (Bellco Glass), cells were centrifuged at 20,000 × g, and the supernatants were used for immunoprecipitation with agarose-conjugated anti-FLAG antibody (M2, Sigma), followed by Western blot analysis with either peroxidase-conjugated anti-HA antibody (F-10, Santa Cruz Biotechnology) or peroxidase-conjugated anti-FLAG antibody.

Immunocytochemistry. COS-7 and SH-SY5Y cells were transiently transfected as described in Results and cultured in glass chamber slides (Becton Dickinson) for 24 h. Cells were fixed in 4% paraformaldehyde in PBS for 20 min, washed with PBS three times, and permeabilized with 0.5% Triton X-100 in PBS for 10 min. After washing with PBS again and blocking with 5% BSA for 20 min, cells were incubated with the indicated antibodies diluted in 1% BSA at room temperature for 1 h and washed five times with PBS. For nuclear staining, cells were incubated with 10 μM DAPI for 1 min. Cells were washed five times with PBS and mounted with antifade mounting material (ProLong, Molecular Probes) under a coverglass and analyzed under a fluorescence microscope (Axiovert 200, Zeiss). For quantification of cells having cytosolic Daxx, 10 microscopic fields, each including 10–50 cells, were randomly selected, and the percentage was counted among cells that were cotransfected as described.

Results

DJ-1 Protects Against Oxidative Stress. Based on loss of function mutations in DJ-1, we hypothesized that this protein serves as a protector of dopaminergic neurons against certain insults commonly studied in PD models. SH-SY5Y dopaminergic neuroblastoma cells were engineered to overexpress full-length FLAG-tagged DJ-1. Western blot analysis of total cell lysates, using both anti-FLAG and a human DJ-1 antibody, confirmed overexpression of DJ-1-engineered cells, compared with empty vector-transfected cells (data not shown). To determine whether overexpression of DJ-1 affects cell viability against oxidative stress conditions, the engineered cells were incubated in the presence of H2O2 (Fig. 1A), dopamine (Fig. 1B), or the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) (Fig. 1C). Exposure of empty vector-transfected cells to these agents resulted in decreased cell viability in a dose-dependent manner, confirming our earlier observations with native SH-SY5Y cells (19). However, DJ-1-overexpressing cells fared significantly better than their control vector-transfected counterparts at the concentration ranges of 200–300 μM H2O2, 100–300 μM dopamine, and 2–5 mM MPP+ (Fig. 1 AC). These results suggest that DJ-1 protects cells against oxidative stress, consistent with recent reports (1013).

Fig. 1.
DJ-1 protects cells against oxidative stress. (AC) SH-SY5Y cells engineered to express FLAG-DJ-1 (filled bars) or transfected with an empty vector (open bars) were challenged with the indicated agents and concentrations for 24 h. Cell survival ...

The impact of the pathogenic L166P mutation in DJ-1 on cell vulnerability was studied next. To obviate potential subtle changes in the genome associated with stable transfections used above, we also tested the cytoprotective properties of DJ-1 in the context of transient expression. SH-SY5Y cells were transfected with FLAG-tagged wild-type or L166P mutant DJ-1, and viability was assessed as described in Materials and Methods. Upon exposure to 0.2 mM H2O2, wild-type DJ-1 expression protected cells by ≈40% (Fig. 1D). On the other hand, DJ-1 carrying the pathogenic mutation L166P was totally ineffective in protecting cells against H2O2, as expected from the autosomal recessive transmission of this point mutation. Similar loss of function by the L166P mutation has been reported (11, 13). It should be pointed out that our data do not show exaggeration of oxidant-induced cell death because of L166P mutant transgene expression and, therefore, do not support a dominant effect as suggested recently (11).

The cytoprotective role of DJ-1 in MPP+-induced cell death was further investigated by using siRNA against DJ-1. Wild-type siRNA significantly reduced DJ-1 expression in a concentration-dependent manner, whereas mutant siRNA did not (Fig. 1E). Under these conditions, MPP+-induced death in siRNA-transfected cells was accelerated with a profile that correlated with the concentration of siRNA and with the decrease in DJ-1 expression (Fig. 1F). On the other hand, mutant siRNA had little effect in aggravating MPP+-induced cell death (data not shown). This result further confirms that DJ-1 confers protection against MPP+-induced cell death.

To examine the extent of the antioxidant activity of DJ-1, intracellular levels of H2O2 were compared between DJ-1 overexpressing and empty vector-transfected SH-SY5Y cells challenged with H2O2 for 1 h (Fig. 2). As expected, H2O2 levels increased in a concentration-dependent manner with the addition of exogenous H2O2 in both cell lines. But DJ-1 overexpression was associated with lower H2O2 levels even in the absence of exogenous H2O2, consistent with an antioxidant activity of DJ-1. However, DJ-1 could reduce H2O2 levels only by ≈20% even with 300 μM H2O2 treatment, which does not fully account for its 5-fold cytoprotection against the same concentration of H2O2 (Fig. 1 A). This observation implies that DJ-1 exerts its protective function through other mechanism(s).

Fig. 2.
DJ-1 inhibits the intracellular accumulation of H2O2. SH-SY5Y cells overexpressing FLAG-DJ-1 (filled bars) or empty vector-transfected (open bars) were treated with H2O2 at the indicated concentrations for 1 h. Intracellular H2O2 was measured by 2′,7′-dichlorofluorescein ...

DJ-1 Interacts with Daxx. To study the molecular mechanism of DJ-1-mediated protection against oxidative stress-induced cell death, we used the yeast two-hybrid screen to identify proteins that interact with DJ-1. The prey consisted of components of a yeast expression library prepared from cDNAs of the human adult brain. Our screen initially isolated 86 specific positive clones, which represented five distinct proteins (Fig. 3A). One of these isolated clones encode DJ-1 itself, a finding consistent with reports that DJ-1 may act as a homodimer (7). Other positive clones that were found to interact with DJ-1 are small ubiquitin-like modifier (SUMO) 1, SUMO-activating enzyme Uba2, and SUMO-conjugating enzyme Ubc9, which are components of the sumoylation system. In line with these results, DJ-1 has been reported to be sumoylated at lysine-130 (15). These findings validate the quality and specificity of our screening method and data. Another notable DJ-1-interacting clone was a protein corresponding to the carboxyl terminus (amino acids 638–739) of human Daxx, which had previously been shown to bind to the intracellular death domain of the Fas receptor and to mediate the activation of c-Jun N-terminal kinase and p38 kinases (20, 21).

Fig. 3.
DJ-1 interacts with Daxx. (A) Interaction in yeast between DJ-1 and clones isolated from the yeast two-hybrid screen. The strength of the interaction with the specific clones as well as with two negative controls (α-synuclein and pYESTrp) was ...

To validate our data from yeast, the binding between DJ-1 and Daxx was tested in COS-7 cells. After transfection with FLAG-DJ-1 and HA-Daxx, complexes were isolated by immunoprecipitation with anti-FLAG and analyzed by Western blotting with anti-HA. This experiment verified that DJ-1 and Daxx interact in mammalian cells (Fig. 3B). Next, we assessed the interaction between Daxx and the pathogenic L166P mutant form of DJ-1. As reported in refs. 22 and 23, the steady-state level of this mutant was lower than its wild-type counterpart because of instability of the mutant protein, which could result in an apparently weaker interaction with Daxx. To circumvent this confounding effect, we transfected the cells with increased amounts of the L166P construct to attain an expression level comparable to that of wild-type DJ-1 (Fig. 3B Bottom). Despite similar amounts of wild-type and L166P DJ-1, the interaction of Daxx to the mutant was much weaker than to wild-type DJ-1 (Fig. 3B Top). This result indicates that the impaired binding of L166P mutant DJ-1 to Daxx is due not to its lower expression level but rather to its conformational change imposed by the mutation.

To complement our finding of DJ-1/Daxx interaction detected by immunoprecipitation, we assessed the subcellular colocalization of these two protein partners by using fluorescent immunocytochemistry after transfecting COS-7 cells with EGFP-DJ-1 and HA-Daxx (Fig. 3C). These studies demonstrated that Daxx is localized mainly in the nucleus, whereas DJ-1 is in both the nucleus and cytoplasm. Therefore, colocalization of these two protein partners occurs in the nucleus.

DJ-1 Protects Against Daxx/ASK1-Induced Cell Death. Daxx interacts with ASK1, causing activation of this kinase, which subsequently promotes cell death (21). Daxx activates ASK1 by relieving an inhibitory intramolecular interaction between the N and C termini of the kinase, allowing it to oligomerize and become activated (21). Therefore, we hypothesized that DJ-1 could hamper the interaction between Daxx and ASK1 by recruiting Daxx, thereby inhibiting ASK1 activation and cell death. First, we tested the effect of DJ-1 on cell death caused by the coexpression of Daxx and ASK1 in SH-SY5Y cells. After transfection with expression vectors of these molecules and with GFP expression plasmid pEGFP-C1 (Clontech), cell death was assayed by using EthD-1 as described in Materials and Methods. Cotransfection with both Daxx and ASK1 resulted in the death of 25% of transfected cells (Fig. 4A), whereas expression of either Daxx or ASK1 alone had no significant impact. Compared with the cell death associated with the combined expression of Daxx and ASK1, the coexpression of wild-type DJ-1 significantly reduced cell death (Fig. 4A). On the other hand, coexpression of the disease-causing L166P mutant DJ-1 was unable to protect cells against this death-signaling pathway. These observations indicate that wild-type but not mutant DJ-1 protects against Daxx/ASK1-induced cell death. Of note is the observation that L166P DJ-1 did not aggravate cell death induced by Daxx/ASK1, once again negating the suggestion that it might act as a dominant negative mutant.

Fig. 4.
DJ-1 protects against Daxx/ASK1-induced cell death and inhibits ASK1 activation. (A) Wild-type but not L166P mutant DJ-1 protects against Daxx/ASK1-induced cell death. SH-SY5Y cells were transfected with the indicated expression vectors, and 24 h later, ...

To determine the effect of DJ-1 on ASK1 activation, COS-7 cells were transfected with Myc-ASK1, HA-Daxx, and FLAG-DJ-1 (wild-type or mutant form). ASK1 was immunoprecipitated with anti-Myc (9E10), followed by in vitro kinase assay. We found that ASK1 activity was potently increased by the coexpression of Daxx, as reported in ref. 21. Notably, the presence of wild-type DJ-1 markedly inhibited Daxx-induced ASK1 activation, whereas the L166P mutant was incapable of repressing the activation of this kinase (Fig. 4B). Collectively, these observations suggest that the cytoprotective effects of DJ-1 may be exerted through inhibition of ASK1 activity.

In addition to Fas-induced, Daxx-mediated activation (21, 24), ASK1 is activated by oxidative stress (25), one of the main suspected culprits in PD pathogenesis (26). As a result, embryonic fibroblasts from ASK1-null mice are resistant to H2O2-induced apoptosis (27). To determine the role of DJ-1 in H2O2-induced ASK1 activation, COS-7 cells were transfected with Myc-ASK1 and FLAG-DJ-1 (wild-type or mutant) and then challenged with 0.5 mM H2O2 for 30 min (Fig. 4C). Treatment with H2O2 markedly increased ASK1 activity as expected. The expression of wild-type DJ-1 significantly reduced H2O2-induced ASK1 activation, whereas the L166P mutant failed to do so. This result suggests that the protective effect of DJ-1 against H2O2-induced cell death (Fig. 1 A and D) is, at least in part, due to its ability to prevent ASK1 activation.

The interaction between Daxx and ASK1 is essential for ASK1 activation and subsequent cell death (21). In addition, our data show that DJ-1 binds to Daxx and prevents Daxx/ASK1-induced apoptosis (Figs. (Figs.33 and 4 A and B). Thus, we raised the possibility that DJ-1 could interfere with the interaction between Daxx and ASK1. To address this question, COS-7 cells were transfected with HA-Daxx plus Myc-ASK1 in the absence or presence of FLAG-DJ-1 (Fig. 4D). Cell lysates were incubated with anti-HA antibody for immunoprecipitation and processed for Western blot analysis with anti-Myc antibody to detect the interaction between HA-Daxx and Myc-ASK1. The presence of wild-type DJ-1 expression vector impaired the interaction between Daxx and ASK-1. On the other hand, the L166P mutant, which was expressed at a level comparable to that of the wild-type protein (Fig. 4D Bottom) by transfecting three times as much plasmid, failed to block the binding between Daxx and ASK1 (Fig. 4D Top). Collectively, these findings suggest that loss of the cytoprotective function of L166P DJ-1 may relate to alterations in its interaction with the death protein Daxx and, consequently, its inability to interfere with the interaction between Daxx and the effector kinase ASK1.

We also tested whether reduced expression of DJ-1 by siRNA renders cells more sensitive to oxidative stress through exaggerated ASK1 activation. SH-SY5Y cells were transiently transfected with siRNA against either DJ-1 or mutant DJ-1 as shown in Fig. 1E, followed by H2O2 treatment for 30 min. Compared with the degree of ASK1 activation in mutant siRNA-transfected cells as control, this kinase was activated to a significantly greater extent in wild-type siRNA-transfected cells (Fig. 4E), confirming that DJ-1 represses ASK1 activation.

DJ-1 Sequesters Daxx in the Nucleus. Daxx is localized mainly to the nucleus of an unstressed cell, particularly in discrete nuclear structures known as promyelocytic leukemia (PML) oncogenic domains, whereas ASK1 is localized in the cytoplasm (24, 28). In cell-death signaling, Daxx translocates to the cytoplasm, a step required before its interaction with ASK1 in response to stress and ASK1 overexpression (24, 28). In SH-SY5Y cells transfected with HA-Daxx alone, ≈2% manifested cytoplasmic HA-Daxx, whereas this proportion jumped to 15% when ASK1 was coexpressed (Fig. 5 A, D, and R). The intracellular distribution of wild-type DJ-1 is ubiquitous, including in the cytoplasm and nucleus as shown in Fig. 3C (22). The L166P mutant has been reported to be localized in mitochondria (22, 23). As part of the inhibitory effect of DJ-1 on Daxx/ASK1-mediated cell death, we hypothesized that DJ-1 binds to Daxx and sequesters it in the nucleus and therefore prevents it from translocating to the cytoplasm and from interacting with and activating ASK1. To determine the effect of DJ-1 on ASK1-induced translocation of Daxx from the nucleus to the cytoplasm, cells were cotransfected with HA-Daxx, Myc-ASK1, and FLAG-DJ-1. Expression of wild-type DJ-1 significantly blocked the translocation of Daxx, with ≈5% of the cells manifesting cytoplasmic Daxx (Fig. 5 G, M, and R). However, expression of L166P mutant DJ-1 failed to do the same, with ≈13% manifesting Daxx in the cytoplasm (Fig. 5 J, P, and R). In addition, based on observations above indicating that DJ-1 inhibits H2O2-induced ASK1 activation and cell death (Figs. 1 A and D and 4 C and E), we tested whether DJ-1 hinders the translocation of Daxx to the cytoplasm upon exposure to H2O2 as well. Challenging HA-Daxx-transfected cells with H2O2 increased the proportion of cells having cytosolic Daxx up to ≈24% (Fig. 5S). Expression of wild-type DJ-1 significantly diminished the translocation of Daxx down to ≈10% of the cells, whereas L166P mutant DJ-1 was ineffective in this regard, with 26% of the cells showing cytosolic Daxx. The results of this experiment suggest that the protective role of DJ-1 in H2O2-induced ASK1 activation and cell death is mediated, in part, by preventing Daxx from translocating to the cytoplasm.

Fig. 5.
DJ-1 inhibits the translocation of Daxx to the cytosol. SH-SY5Y cells were transfected with the indicated plasmids, and Daxx was stained red (A, D, G, J, M, and P), ASK1 was stained green (C, F, and I), and DJ-1 also was stained green (L and O). Nuclei ...

Discussion

The present study demonstrates that DJ-1 protects cells against oxidative stress, but that its ability to quench reactive oxygen species is insufficient to account for its overall cytoprotective function. In search for additional mechanisms for this function, we report here that DJ-1 is a potent inhibitor of the Daxx/ASK1 cell-death signaling pathway, a hitherto unexplored pathway in dopaminergic neurons or in PD. We find that wild-type DJ-1 interacts with Daxx, sequestering it in the nucleus and preventing it from gaining access to the cytoplasm, from interacting with its effector kinase ASK1, from activating this kinase, and hence, from cell death. The pathogenic L166P mutant form of DJ-1 is impotent in each of these steps. These observations collectively point to a specific pathway by which DJ-1 functions as a survival factor.

The subcellular localization of Daxx appears to be a crucial factor in its death signaling function. Daxx was originally identified as an adaptor protein of the Fas receptor mediating apoptosis (20). Fas activation induces Daxx to interact with ASK1, leading to activation of this kinase. However, the fact that Daxx is a nuclear protein whereas ASK1 is cytosolic had been a major point of discrepancy. Several studies have since found that a significant portion of Daxx relocalizes from the nucleus to the cytosol upon various stresses, including Fas activation, glucose deprivation, H2O2 treatment, and ASK1 overexpression (24, 28, 29), thus providing a rational basis for its interaction with ASK1. We also confirmed the relocalization of Daxx upon ASK1 overexpression. Under this condition, we found that DJ-1 effectively prevents the egress of Daxx from the nucleus and therefore impairs its interaction with ASK1, decreases activation of this kinase, and reduces cell death. Thus, the regulated localization of Daxx appears to be a powerful mechanism controlling cell death vs. survival. It would be of interest to determine whether Daxx is aberrantly localized to the cytosol in the brains of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice and in PD-affected brains, where indices of oxidative stress are known to be elevated (26).

In addition to its proapoptotic role in the cytosol, Daxx is a nuclear protein that usually represses transcription (30) by means of direct protein–protein interactions with several specific transcription factors, including Pax3 and ETS1 (31, 32). In addition, Daxx promotes cellular sensitivity to Fas from a nuclear location by modulating the transcription of genes involved in Fas-induced cell death (33). DJ-1 binds to transcriptional repressors, such as protein inhibitor of activated STAT (PIAS)xα and DJ-1-binding protein, and antagonizes their repressive function (14, 15). Therefore, it is of interest to study whether DJ-1 modulates transcription by virtue of binding to the transcription repressor Daxx and how this relates to its cytoprotective function.

The role of ASK1 in neurodegenerative diseases is beginning to be recognized. The cytoplasmic domain of amyloid precursor protein dimerizes and forms a complex with ASK1 by means of the c-Jun N-terminal kinase (JNK)-interacting protein JIP-1b, causing sustained ASK1/JNK-mediated neurotoxic signal (34). Primary neurons derived from ASK1-/- mice are resistant to endoplasmic reticulum stress-, proteasome dysfunction-, and polyglutamine-induced cell death (35). ASK1 also has been found to be involved in the mechanism of seizure-induced neuronal death (36). The participation of ASK1 in the neuronal degeneration in PD-affected brains is yet to be explored. However, based on the foregoing observations, it appears likely that ASK1 plays a key role in the demise of dopaminergic neurons because of the clear involvement of this kinase in oxidative stress-induced cell death and the excessive oxidative milieu of these neurons. The findings reported here point to a critical role of DJ-1 in keeping this death pathway under control, a function lost by a PD-causing mutation.

Acknowledgments

This work was supported by grants from the American Parkinson Disease Association and the Parkinson's Disease Foundation. M.M.M. is the William Dow Lovett Professor of Neurology.

Notes

Author contributions: E.J. and M.M.M. designed research; E.J., H.T., B.S.J., and X.Z. performed research; H.I. contributed new reagents/analytic tools; E.J. and M.M.M. analyzed data; and E.J. and M.M.M. wrote the paper.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: PD, Parkinson's disease; siRNA, short interfering RNA; HA, hemagglutinin; EthD-1, ethidiumhomodimer1;ASK1, apoptosissignal-regulatingkinase1;MPP+,1-methyl-4-phenylpyridinium.

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