DJ‐1‐binding compounds prevent oxidative stress‐induced cell death and movement defect in Parkinson’s disease model rats

Abstract

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.

Oxidative Status of DJ-1-dependent Activation of Dopamine Synthesis through Interaction of Tyrosine Hydroxylase and 4-Dihydroxy-l-phenylalanine (l-DOPA) Decarboxylase with DJ-1

“…In 19), FLAG-DJ-1, endogenous DJ-1, and ␤-actin were analyzed as described in the legends of Fig. 1, E and F, and Fig.…”

“…and DDC-To examine the effects of DJ-1 on expression and activity of TH and DDC, human neuroblastoma SH-SY5Y cells, in which the DJ-1 gene was knocked down, were established by transfection of a vector containing short hairpin RNA targeting human DJ-1, as described previously (19), and were named "DJ-1 knockdown cells" (19). SH-SY5Y cells harboring the vector without short hairpin RNA were also established and named "Vector cells."…”

“…An aliquot of these proteins was analyzed by Western blotting with anti-TH and anti-DDC antibodies (I), and the other aliquots were used to measure TH and DDC activities as described under "Experimental Procedures" (J and K, respectively). L, proteins in reaction mixtures for TH activity as shown in G were analyzed by Western blotting with anti-phosphorylated TH with a serine residue at amino acid number 19 (pTH ser 19), anti-TH, anti-DJ-1, and anti-␤-actin antibodies. Significance: *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001 compared with an amount of "lysate ϩ substrate," and #, p Ͻ 0.05; ##, p Ͻ 0.01; ###, p Ͻ 0.001 compared with the amount of "lysate ϩ substrate ϩ GST."…”

Common Mechanisms of Onset of Cancer and Neurodegenerative Diseases

“…These compounds inhibit excess oxidation of C106 of DJ-1 and maintain the reduced form DJ-1, thereby inhibiting oxidative stress-induced neuronal cell death and mitigating locomotion defect in mouse and rat PD models concomitantly with increased TH and mitochondrial complex I activities. These compounds are able to penetrate the BBB 192,195,196) and are also effective in stroke model rats. 197) Protective activities of DJ-1-binding compounds are lost in DJ-1-knockdown or -knockout cells and in DJ-1-knockout mice, indicating the DJ-1-dependency of compounds.…”

“…We first clarified the X-ray crystal structure of DJ-1 and found that there is a pocket around C106 of DJ-1. 108) After in silico screening for DJ-1-binding compounds using a university compound library and Zinc library, various compounds, including compound B/UCP0054278, compound A/ UCP0045037 and compound-23, were obtained [192][193][194][195] (Fig. 7).…”