Parkinson's disease (PD) is caused by dopaminergic cell death, and genetic and environmental factors are thought to affect the onset of PD. These factors lead to oxidative stress, mitochondrial dysfunction and impairment of the protein degradation system, resulting in cell death. Although a precursor of dopamine and inhibitors of dopamine degradation have been used for PD therapy, cell death progresses during treatment. Identification of compounds or proteins that inhibit oxidative stress-induced neuronal cell death is necessary. DJ-1 was first identified by our group as a novel Received October 29, 2007; revised manuscript received February 25, 2008; accepted February 26, 2008. Address correspondence and reprint requests to Hiroyoshi Ariga, PhD, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan. E-mail: hiro@pharm.hokudai.ac.jp 1 Shin Miyazaki and Takashi Yanagida contributed equally to this work.Abbreviations used: 6-OHDA, 6-hydroxydopamine; BBB, bloodbrain barrier; DMEM, Dulbecco's modified Eagle's medium; PD, Parkinson's disease; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMSO, dimethylsulfoxide; QCM, quartz crystal microbalance; ROS, reactive oxygen species; TH, tyrosine hydroxylase. AbstractParkinson's disease (PD) is caused by neuronal cell death. Although a precursor of dopamine and inhibitors of dopamine degradation have been used for PD therapy, cell death progresses during treatment. DJ-1, a causative gene product of a familial form of PD, PARK7, plays roles in transcriptional regulation and anti-oxidative stress, and loss of its function is thought to result in the onset of PD. Superfluous oxidation of cysteine at amino acid 106 (C106) of DJ-1 renders DJ-1 inactive, and such oxidized DJ-1 has been observed in patients with the sporadic form of PD. In this study, we isolated compounds that bind to the region at C106 by a virtual screening. These compounds prevented oxidative stress-induced death of SH-SY5Y cells, embryonic stem cell-derived dopaminergic cells and primary neuronal cells of the ventral mesencephalon, but not that of DJ-1-knockdown cells of SH-SY5Y and NIH3T3 cells, indicating that the effect of the compounds is specific to DJ-1. These compounds inhibited production of reactive oxygen species and restored activities of mitochondrial complex I and tyrosine hydroxylase that had been compromised by oxidative stress. These compounds prevented dopaminergic cell death in the substantia nigra and restored movement abnormality in 6-hydroxyldopamine-injected PD model rats. One mechanism of action of these compounds is prevention of superfluous oxidation of DJ-1, and the compounds passed through the blood-brain barrier in vitro. Taken together, the results indicate that these compounds should become fundamental drugs for PD therapy.

Parkinson disease (PD) is caused by loss of dopamine, which is synthesized from tyrosine by two enzymes, tyrosine hydroxylase (TH) and 4-dihydroxy-L-phenylalanine decarboxylase (DDC).DJ-1 is a causative gene for the familial form of PD, but little is known about the roles of DJ-1 in dopamine synthesis. In this study, we found that DJ-1 directly bound to TH and DDC and positively regulated their activities in human dopaminergic cells. Mutants of DJ-1 found in PD patients, including heterozygous mutants, lost their activity and worked as dominant-negative forms toward wild-type DJ-1. When cells were treated with H 2 O 2 , 6-hydroxydopamine, or 1-methyl-4-phenylpyridinium, changes in activities of TH and DDC accompanied by oxidation of cysteine 106 of DJ-1 occurred. It was found that DJ-1 possessing Cys-106 with SH and SOH forms was active and that DJ-1 possessing Cys-106 with SO 2 H and SO 3 H forms was inactive in terms of stimulation of TH and DDC activities. These findings indicate an essential role of DJ-1 in dopamine synthesis and contribution of DJ-1 to the sporadic form of PD. Parkinson disease (PD)2 is a neurodegenerative disease that occurs by reduction of the dopamine level through dopaminergic cell death in the substantia nigra. Genetic and environmental factors are thought to be triggers for the onset of PD, but the precise molecular mechanisms are still not known. Dopamine is synthesized by the following two steps: tyrosine is converted to L-DOPA by tyrosine hydroxylase (TH) and then L-DOPA is converted to dopamine by L-dopa decarboxylase (DDC). TH is therefore a key enzyme for dopamine synthesis and is used as a marker for dopaminergic neurons.DJ-1 was first identified by our group as a novel candidate of the oncogene that transformed mouse NIH3T3 cells in cooperation with activated ras (1). Deletion and point (L166P) mutations of DJ-1 have been shown to be responsible for onset of familial Parkinson disease, PARK7 (2), and other homozygous and heterozygous mutations of DJ-1 have been identified in patients with familial or sporadic Parkinson disease (3-6). DJ-1 is a multifunctional protein and plays roles in transcriptional regulation and anti-oxidative stress function, and loss of its functions is thought to lead to the onset of Parkinson disease and cancer. DJ-1 has three cysteine residues at positions 46, 53, and 106 (Cys-46, Cys-53, and Cys-106, respectively), and these cysteine residues are oxidized after cells receive oxidative stress, resulting in scavenging of reactive oxidative species (7-12). Although DJ-1 does not directly bind to DNA, DI-1 acts as a co-activator to activate various transcription factors, including the androgen receptor and p53 tumor suppressor, PSF and Nrf2, by sequestering their inhibitory factors (13-18). The anti-oxidative stress function of DJ-1 is therefore thought to be carried out both by self-oxidation of cysteine residues and by activation of redox-related genes.It has been reported that PSF, a transcription repressor, binds to the promoter region of the TH gene...

Loss-of-function mutation in the DJ-1 gene causes a subset of familial Parkinson disease. The mechanism underlying DJ-1-related selective vulnerability in the dopaminergic pathway is, however, not known. DJ-1 has multiple functions, including transcriptional regulation, and one of transcriptional target genes for DJ-1 is the tyrosine hydroxylase (TH) gene, the product of which is a key enzyme for dopamine biosynthesis. It has been reported that DJ-1 is a neuroprotective transcriptional co-activator that sequesters a transcriptional co-repressor polypyrimidine tract-binding protein-associated splicing factor (PSF) from the TH gene promoter. In this study, we found that knockdown of human DJ-1 by small interference RNA in human dopaminergic cell lines attenuated TH gene expression and 4-dihydroxy-l-phenylalanine production but that knockdown or knock-out of mouse DJ-1 in mouse cell lines or in mice did not affect such expression and TH activity. In reporter assays using the human TH gene promoter linked to the luciferase gene, stimulation of TH promoter activity was observed in human cells, but not mouse cells, that had been transfected with DJ-1. Although human DJ-1 and mouse DJ-1 were associated either with human or with mouse PSF, TH promoter activity inhibited by PSF was restored by human DJ-1 but not by mouse DJ-1. Chromatin immunoprecipitation assays revealed that the complex of PSF with DJ-1 bound to the human but not the mouse TH gene promoter. These results suggest a novel species-specific transcriptional regulation of the TH promoter by DJ-1 and one of the mechanisms for no reduction of TH in DJ-1-knock-out mice.

DJ-1 is secreted into the serum and plasma of patients with various diseases. In this study, DJ-1 was found to be secreted into culture media of various cells and the amount of wild-type DJ-1 secreted was two-fold greater than that of mutant DJ-1 of cysteine at 106 (C106). Furthermore, the oxidative status of more than 90% of the DJ-1 secreted from HeLa cells was SOH and SO 2 H forms of C106. A portion of DJ-1 in cells was localized in microdomains of the membrane. These findings suggest that DJ-1 is secreted through microdomains and that oxidation of DJ-1 at C106 facilitates the secretion.