The idea that mutations in the DJ-1 gene cause early onset Parkinson disease is unquestioned, but just how loss of DJ-1 function leads to destruction of dopamine neurons has been a query without an answer so far. Mounting evidence points to DJ-1’s role as protector of neurons undergoing oxidative stress, but how does the protein prevent cell death? Now, M. Maral Mouradian and colleagues at the Robert Wood Johnson Medical School in New Jersey and in Japan have an answer. In their paper published in last week’s PNAS online, they report that DJ-1 blocks a specific apoptotic pathway triggered by oxidative stress. By sequestering the death protein Daxx, DJ-1 prevents the activation of the pro-apoptotic kinase ASK-1. A disease-causing mutant of DJ-1 (L166P) fails to bind Daxx, block ASK-1 activation, or protect cells from oxidative stress, suggesting that the withdrawal of DJ-1’s survival signal could underlie the loss of neurons observed in Parkinson disease.

The search for DJ-1’s protective mechanism has uncovered putative functions ranging from free radical scavenger and protein chaperone to transcriptional regulator (see ARF related news story). In the latest foray, first author Eunsung Junn began by confirming that DJ-1 could protect cells from oxidative stress. Consistent with other recent work (see ARF related news story), SH-SY5Y dopaminergic neuroblastoma cells engineered to overexpress DJ-1 were less sensitive to the toxic effects of hydrogen peroxide, dopamine, or MPP+, while RNAi knockdown of endogenous DJ-1 made the cells more sensitive. A PD mutant of DJ-1, L166P, did not protect the cells. To test whether DJ-1 had a direct antioxidant effect, the researchers measured peroxide in cells overexpressing DJ-1. While peroxide decreased 20 percent, cell death decreased fivefold, leading them to look for other explanations for DJ-1’s protective power.

Turning to yeast 2-hybrid assay to identify DJ-1 partners from a human adult brain cDNA library, the investigators found five candidate proteins. Four were no surprise—one was DJ-1 itself and three were proteins involved in sumoylation, a ubiquitin-related posttranslational modification previously reported for DJ-1. But the fifth clone was Daxx, a death protein known to bind to the intracellular death domain of Fas receptor and mediate activation of downstream pro-apoptotic kinases.

Cotransfection and coprecipitation experiments showed that Daxx and DJ-1 interacted in cells, as well. In SH-SY5Y cells, expression of DJ-1 could prevent cell death induced by introduction of Daxx plus its downstream target, the apoptosis-signal regulating kinase-1 (ASK-1). ASK-1 activation by peroxide was blocked by expression of DJ-1, and introduction of DJ-1 RNAi enhanced activation of ASK-1. These results suggested that the protective effect of DJ-1 against peroxide-induced death is at least in part due to preventing ASK-1 activation.

How, exactly, does DJ-1 block ASK-1 activation? Daxx is known to activate the kinase via a direct interaction, and the researchers were able to show that DJ-1 interfered with the association of ASK-1 and the cell death protein. In fact, Daxx normally resides in the nucleus, and during oxidative insult it moves to the cytosol where it binds and activates the kinase. For example, in Daxx-transfected SH-SY5Y cells treated with peroxide, up to 24 percent of the cells showed cytoplasmic Daxx. But the authors found that if the cells also expressed DJ-1, only about 10 percent had detectable Daxx in their cytoplasm. With the cell death protein held in the nucleus, ASK-1 languished, unactivated, in the cytosol.

Through all the experiments, the L166P DJ-1 mutant demonstrated a loss-of-function phenotype. The mutant DJ-1 was ineffective at protecting cells, interacting with Daxx, or influencing ASK-1 activity, consistent with the Daxx/ASK-1 pathway’s contribution to PD pathology. While the Daxx/ASK-1 apoptotic pathway has been implicated in the death of motor neurons from ALS mice (see ARF related news story) and in response to polyglutamine-repeat containing proteins (Nishitoh et al., 2002), these proteins had not been previously implicated in DA neuron death or PD. The next step will be to look at Daxx in brains of MPTP-treated mice or PD-affected brains, where oxidative stress markers are elevated.—Pat McCaffrey

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  1. The observation that DJ-1 is capable of protecting cells against oxidant-induced cell death has been confirmed in several laboratories without a very clear mechanism emerging for how this would happen. Showing that simple scavenging of radicals is not likely to account for neuroprotection, as Junn et al. do in this study, is important. It has never seemed likely to me that a protein like DJ-1 provides very much antioxidant capacity in a cell full of low molecular weight thiols. Junn et al. show that the previous data from Ariga showing that DJ-1 decreases intracellular hydrogen peroxide is correct, but argue that there must be another protective mechanism as the decrease in peroxide levels is smaller than the percentile suppression of cell death. They have gone on to show that DJ-1 interacts with, amongst other proteins, Daxx, and suggest that this mechanism accounts for neuroprotection via an ASK1 pathway. Although ASK1 is named for its effects on apoptosis, I wonder if DJ-1’s protective ability is on apoptosis per se or whether it is broader than that. Yokota et al. have shown that DJ-1 is not a suppressor of staurosporine-induced apoptosis. Perhaps DJ-1 requires activation, such as by oxidation, before it can protect cells. Also, at least in my lab, MPP+ doesn’t produce a strict apoptotic cell death even though DJ-1 is protective. It is interesting that although Daxx and ASK1 usually work through classical apoptosis pathways (Song and Lee, 2004), ASK1 can also activate cell death in a fashion that doesn’t include many of the hallmarks of nuclear apoptosis (Zhang et al., 2004).

    References:

    . Down regulation of DJ-1 enhances cell death by oxidative stress, ER stress, and proteasome inhibition. Biochem Biophys Res Commun. 2003 Dec 26;312(4):1342-8. PubMed.

    . Daxx deletion mutant (amino acids 501-625)-induced apoptosis occurs through the JNK/p38-Bax-dependent mitochondrial pathway. J Cell Biochem. 2004 Aug 15;92(6):1257-70. PubMed.

    . Thioredoxin-2 inhibits mitochondria-located ASK1-mediated apoptosis in a JNK-independent manner. Circ Res. 2004 Jun 11;94(11):1483-91. PubMed.

  2. The recent finding from Junn and the Mouradian group on DJ-1 interacting with Daxx is very interesting. First, by using the two hybridization system, Junn et al. found that DJ-1 can bind Daxx, which is a component of the Fas death receptor signal transduction pathway. Moreover, they found that DJ-1 protects cells not only from free radicals, but also against Daxx/ASK-1-induced cell death. This study provides important evidence to elucidate of functional mechanisms of DJ-1 antioxidation. These studies are done in vitro. It will be interesting to examine whether the functional relationship between DJ-1 and Daxx will retain the same pattern in vivo. Furthermore, more studies need to investigate why the DJ-1 protective signal transduction pathway mainly occurs in the dopaminergic neuronal system, since DJ-1 or Daxx are widely expressed not only in the brain, but also in the peripheral nervous system, and not only in the dopamine system, but also in many other systems in the brain. Lastly, one important function of DJ-1 is considered to depend on macrocomplexes in normal brains, since once DJ-1 becomes a monomer or oligomer, it may lose its protective function. However, all of the studies Junn et al. reported were conducted in vitro and DJ-1 seems to interact with other molecules, i.e., Daxx as a monomer. DJ-1 functions and roles in neuroprotection need further exploration, discussion, and interpretation.

References

News Citations

  1. DJ Chaperones α-Synuclein—Offers Cell Model of Parkinson Disease
  2. Protection Against Parkinson’s—How the DJ Changes Station
  3. A Cell-Specific Death Program Claims Motoneurons

Paper Citations

  1. . ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Genes Dev. 2002 Jun 1;16(11):1345-55. PubMed.

Further Reading

Primary Papers

  1. . Interaction of DJ-1 with Daxx inhibits apoptosis signal-regulating kinase 1 activity and cell death. Proc Natl Acad Sci U S A. 2005 Jul 5;102(27):9691-6. PubMed.