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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- Nishitoh H, Matsuzawa A, Tobiume K, Saegusa K, Takeda K, Inoue K, Hori S, Kakizuka A, Ichijo H. 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.
- Junn E, Taniguchi H, Jeong BS, Zhao X, Ichijo H, Mouradian MM. 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.