Skip Navigation


Human Molecular Genetics Advance Access originally published online on September 6, 2007
Human Molecular Genetics 2007 16(23):2900-2910; doi:10.1093/hmg/ddm249
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
16/23/2900    most recent
ddm249v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Yang, W.
Right arrow Articles by Kang, U. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, W.
Right arrow Articles by Kang, U. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Paraquat induces dopaminergic dysfunction and proteasome impairment in DJ-1-deficient mice

Wonsuk Yang1,{dagger}, Linan Chen2,{dagger}, Yunmin Ding1, Xiaoxi Zhuang2 and Un Jung Kang1,*

1 Department of Neurology and 2 Department of Neurobiology, The University of Chicago, IL, USA

* To whom correspondence should be addressed at: Department of Neurology, The University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, USA. Tel: +1 7737026389; Fax: +1 7737026538; Email: unkang{at}uchicago.edu

Received July 19, 2007; Accepted August 24, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Parkinson’s disease (PD) may be caused by a complex interaction of environmental insults and genetic susceptibilities. Previous studies of DJ-1-deficient mice have noted dopaminergic dysfunction mainly in older mice. To simulate the interaction of genetic factors and environmental factors, we treated DJ-1-deficient mice with paraquat. Even in relatively young mice, this combination produced dopamine loss and motor dysfunction. To determine the potential mechanism for the dopaminergic dysfunction, we investigated the proteasome function and ubiquitinated protein levels. DJ-1-deficient mice treated with paraquat showed decreased proteasome activities and increased ubiquitinated protein levels. To further investigate the mechanism of proteasome dysfunction, ATP levels and subunit protein levels of 19S ATPase Rpt6 and 20S ß5 were measured and noted to be decreased in the ventral midbrain, but not in the striatum. Finally, a transcription factor, Nrf2 that has been previously shown to be regulated by DJ-1 and to regulate 20S ß5 levels was decreased. These pathologies were not observed in brain regions of normal mice treated with paraquat. In conclusion, this study raises the possibility that environmental and genetic factors might cooperatively involve the mechanisms underlying proteasome impairment in PD brains.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Parkinson’s disease (PD) is the second most common idiopathic progressive neurodegenerative disorder and affects 1–2% of the population over the age of 65, further increasing in incidence at older ages (1). Most cases of PD are sporadic and the familial forms of PD account for less than 10% of total cases (2). PD is pathologically characterized by a relatively selective loss (50–70%) of dopaminergic neurons in the A9 area of human brain known as the substantia nigra pars compacta (SNpc) and a dramatic reduction of striatal dopamine levels (3). An additional important pathological feature of PD is the presence of intracytoplasmic inclusions called Lewy bodies in the neuronal cell body and Lewy neurites in neuronal processes of the remaining dopaminergic neurons (4).

The etiology of PD is not completely defined, although contributions of both environmental factors and genetic susceptibility have been demonstrated. In most cases, combination of these factors is suspected to play a role in the pathogenesis. Exposure to pesticides has been most consistently implicated as an environmental factor. In particular, epidemiologic reports suggest that the exposure to paraquat (methyl viologen, 1, 1'-dimethyl-4, 4' bipyridium dichloride), a bipyridyl herbicide widely used in agriculture is associated with the development of PD in humans (5–7). This hypothesis is supported by animal studies demonstrating that paraquat reproduces most of the hallmarks of dopaminergic pathogenesis of PD (8–10). Paraquat decreases dopamine levels and the number of dopamine neurons in the SNpc. Furthermore, paraquat increases protein levels of {alpha}-synuclein and the incidence of {alpha}-synuclein-positive aggregates in the ventral midbrain (VMB) or dopaminergic neurons of mice, respectively (11).

DJ-1 is a highly conserved 189 amino acid protein, which belongs to the ThiJ/PfpI protein superfamily (12) and its mutations have been associated with an autosomal recessive form of early-onset PD (13). DJ-1 is ubiquitously expressed in many tissues including the brain, where it is present both in neurons and glial cells (14,15). The functions of DJ-1 are not completely characterized, but recent studies suggest that it might serve as antioxidant, redox-sensitive molecular chaperone and transcription regulator (16–19). Importantly, knockdown or deletion of DJ-1 increases susceptibility to proteasome inhibition in cultured cells (20,21). In addition, the deletion of DJ-1 induces the aggregation of {alpha}-synuclein, whereas the overexpression of DJ-1 inhibits the formation of {alpha}-synuclein aggregates in cultured cells (22,23). Thus, these studies suggest that DJ-1 may affect protein clearance pathway including the proteasomal pathway.

The proteasome is a large multi-catalytic proteinase complex found in the nucleus and cytoplasm of eukaryotic cells (24,25). It degrades damaged, misfolded, mutated and unfolded intracellular proteins as the major protein quality control system of eukaryotes (26). The impairment of proteasome function might lead to aberrant protein aggregation or accumulation, ultimately resulting in cell death (27,28). The decrease of proteasome activity is reported in PD brains (29,30). In addition, low levels of 20S {alpha} and 19S subunits are observed in dopaminergic neurons of PD brains (31). These evidences have implied proteasome impairment in the pathogenesis of PD (32).

Therefore, we addressed the combined effect of genetic and environmental factors in the pathogenesis of PD by using DJ-1-deficient mice treated with paraquat. Having found motor dysfunction and dopamine deficit in these animals, we focused the investigation on abnormal protein degradation pathway as a potential mechanism for the dopaminergic dysfunction. We examined proteasome activities, proteasome subunits and ubiquitinated protein levels, as well as factors that regulate the proteasome such as ATP and an oxidation-sensitive transcription factor Nrf2 (nuclear factor erythroid 2-related factor).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Paraquat impaired the motor performance in DJ-1-deficient mice
We have previously found that motor performance deteriorates in aged DJ-1-deficient mice compared to wild-type mice, but the difference between the genotypes was not evident in younger mice (33). Consistent with our previous data (33), rotarod and open field test performances were similar in DJ-1-deficient mice and wild-type mice at the young age (6 months) tested. Interestingly, rotarod performance was impaired only in DJ-1-deficient mice treated with parquat (Fig. 1A), as measured by the duration that mice can stay on a rotating rod (P < 0.05, n = 8). In contrast, wild-type mice were not affected by the current paraquat treatment paradigm. We also examined mice using the open field test, which showed similar trends of a synergistic effect of paraquat treatment and DJ-1 deficiency, although not at a statistically significant level (P = 0.19 for horizontal movement, Fig. 1B and P = 0.09 for vertical movement, Fig. 1C, n = 8).


Figure 1
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. Effect of paraquat on behavioral performances of DJ-1-deficient mice when compared with wild-type mice. The latency to fall in accelerated Rotarod was measured (A). Horizontal movement (B) and vertical movement (C) in open field were recorded. WS, wild-type mice exposed to saline; WP, wild-type mice exposed to paraquat; DS, DJ-1-deficient mice exposed to saline; DP, DJ-1-deficient mice exposed to paraquat. *P < 0.01 when compared to DJ-1-deficient mice exposed to saline (n = 8, each group).

 
Paraquat decreased striatal dopamine content without dopamine neuron loss in DJ-1-deficient mice
The striatum is the major target region of VMB dopamine neuron and its dopamine content mostly reflects the dopamine vesicular storage in dopamine terminal. We examined how behavioral changes correlate with dopamine content in the striatum and dopamine neuron number in VMB. Consistent with our previous publication (33), striatal dopamine level was higher in DJ-1-deficient mice than in wild-type mice. Interestingly, paraquat treatment decreased dopamine level by 30% in DJ-1-deficient mice (P < 0.05, n = 15). This effect was not seen in wild-type mice with the same paraquat treatment (Fig. 2A). It is of note that this 30% reduction of striatal dopamine in DJ-1 deficient mice reduced the neurotransmitter level similar to that of wild-type mice. Paraquat alone or in combination with manganese ethylenebisdithiocarbamate has been reported to induce behavioral deficit, dopamine depletion and neuronal cell loss in a number of publications and the extent of the loss varies in different studies and with the age of the animals (9,34–36). In our present study, even though significant deficits in rotarod performance and striatal dopamine content were found, the dopamine neuron number in SNpc assessed by unbiased stereological cell counting did not decrease with paraquat treatment (P > 0.05, n = 8, Fig. 2B). Our data suggest that the behavioral and neurochemical consequences manifest before actual dopamine neuron degeneration. The dysfunction of dopamine neurons caused by the synergistic effect of paraquat and DJ-1 deficiency could contribute to these changes.


Figure 2
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. Effects of paraquat on striatal dopamine content and dopamine neuron cell number in DJ-1-deficient and wild-type mice. Dopamine tissue contents in striatum were measured by HPLC (A). Dopamine neuron number in the substantia nigra pars compacta (SNpc) was assessed by unbiased stereological cell counting (B). WS, wild-type mice exposed to saline; WP, wild-type mice exposed to paraquat; DS, DJ-1-deficient mice exposed to saline; DP, DJ-1-deficient mice exposed to paraquat. *P < 0.05 when compared to DJ-1-deficient mice exposed to saline (n = 15, each group).

 
Paraquat decreased proteasome activities in the brain of DJ-1-deficient mice
To understand the cellular dysfunction arising from interaction of the DJ-1 deficiency and paraquat exposure, we examined potential abnormalities in the protein processing machineries. The proteasome complex shows multiple peptidase activities including chymotrypsin-, trypsin- and peptidylglutamyl-peptide hydrolyzing (PGPH)-like activities, which are responsible for protein cleavage at hydrophobic, basic or acidic residues, respectively (37). To examine whether paraquat affects proteasome function in mouse brain, lysates of the striatum and VMB of DJ-1-deficient and wild-type mice treated with saline or paraquat were incubated with each specific fluorogenic substrate to measure chymotrypsin-, trypsin- or PGPH-like activity, respectively (Fig. 3). There was a significant effect of paraquat on proteasome activities (P < 0.005 for treatment effect by two-way ANOVA for all three types of activities), but no overall genotypic effect or interaction between genotype and treatment. Proteasome activities of VMB decreased by ~30% in DJ-1-deficient mice treated with paraquat compared to the saline-treated group (P < 0.05 by post hoc test) (Fig. 3D–F). Proteasome activities in striatal tissue were not significantly altered by paraquat treatment or DJ-1 deficiency (Fig. 3A–C).


Figure 3
View larger version (32K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3. Effects of paraquat on proteasome activities in the brain of DJ-1-deficient and wild-type mice. Chymotrypsin (CHTR: A, D)-, trypsin (TR; B, E)- and peptidylglutamyl-peptide hydrolyzing (PGPH; C, F)-like activities of proteasome were measured in the striatum (A–C) and ventral midbrain (VMB; D–F) from each group. WS, wild-type mice exposed to saline; WP, wild-type mice exposed to paraquat; DS, DJ-1-deficient mice exposed to saline; DP, DJ-1-deficient mice exposed to paraquat. Data are expressed as mean percentage of wild-type mice exposed to saline ± SEM (n = 9, each group). *P < 0.05 when compared to DJ-1-deficient mice exposed to saline.

 
Paraquat increased ubiquitinated protein level in the brain of DJ-1-deficient mice
Ubiquitin plays an important role in tagging unnecessary or damaged proteins for degradation by the proteasome complex (38) and proteasome dysfunction leads to the abnormal accumulation of ubiquitinated protein (39). Thus, experiments were performed to investigate whether paraquat affects the levels of ubiquitinated protein in mouse brain (Fig. 4). Lysates of the striatum and VMB of normal and DJ-1-deficient mice treated with saline or paraquat were run on the gradient (4–20%) Tris–HCl SDS gels and ubiquitinated proteins were detected with a rabbit polyclonal anti-ubiquitin antibody. There were significant effects of paraquat treatment, genotype and their interaction on ubiquitinated protein levels in VMB (P < 0.05 by two-way ANOVA). Whereas ubiquitinated protein levels of VMB were not influenced by paraquat treatment in wild-type mice nor by the absence of DJ-1 without paraquat treatment (Fig. 4C and D), they increased significantly in paraquat-treated DJ-1-deficient mice compared to saline-treated DJ-1-deficient mice (259.3 ± 4.7%, P < 0.05) or paraquat treated wild-type mice (Fig. 4C and D). Ubiquitinated protein levels in the striatum were not influenced by paraquat treatment or DJ-1-deficiency (Fig. 4A and B).


Figure 4
View larger version (49K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4. Effects of paraquat on the levels of ubiquitinated proteins (UBP) in the brain of DJ-1-deficient and wild-type mice. (A) Representative western blot (A, C) and quantification of ubiquitinated proteins (UBP; B, D) in the striatum (A, B) and the VMB (C, D) from each group. WS, wild-type mice exposed to saline; WP, wild-type mice exposed to paraquat; DS, DJ-1-deficient mice exposed to saline; DP, DJ-1-deficient mice exposed to paraquat). Band areas of ubiquitinated proteins were quantitated to determine their levels and normalized to the level of ß-actin. The quantified values of ubiquitinated protein level are expressed as mean percentage of wild-type mice exposed to saline ± SEM (n = 4, each group). *P < 0.05 when compared to DJ-1-deficient mice exposed to saline.

 
Paraquat decreased ATP level in the brain of DJ-1-deficient mice
ATP is required for the binding of ubiquitinated protein to the proteasome 20S {alpha} ring, opening of gate of 20S {alpha} ring, followed by the translocation of unfolded proteins into 20S core by 19S ATPase subunits (40). Therefore, ATP depletion could induce proteasome dysfunction accompanied by the aberrant accumulation of ubiquitinated protein (41,42). Given this possibility, experiments were conducted to examine whether paraquat affects ATP levels in mouse brain (Fig. 5). ATP levels were measured in the lysates of the striatum and VMB of DJ-1-deficient and wild-type mice treated with saline or paraquat. There were significant effects of paraquat treatment on ATP levels in VMB of DJ-1 deficient mice (P < 0.05 by two-way ANOVA). Whereas VMB ATP levels were not influenced by paraquat treatment in wild-type mice or by the absence of DJ-1 without paraquat treatment (Fig. 5A), it decreased significantly in paraquat-treated DJ-1-deficient mice when compared with saline-treated DJ-1-deficient mice (67.6 ± 6.8%, P < 0.05) or paraquat treated wild-type mice (Fig. 5B). ATP levels in the striatum were not influenced by paraquat treatment or DJ-1-deficiency.


Figure 5
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5. Effect of paraquat on ATP levels in the brain of DJ-1-deficient and wild-type mice. ATP levels were determined in the lysates of the striatum (A) and VMB (B) from each group. WS, wild-type mice exposed to saline; WP, wild- type mice exposed to paraquat; DS, DJ-1-deficient mice exposed to saline; DP, DJ-1-deficient mice exposed to paraquat. Data are expressed as mean percentage of wild-type mice exposed to saline ± SEM (n = 9, each group). *P < 0.05 when compared to DJ-1-deficient mice exposed to saline.

 
Paraquat decreased the protein levels of proteasomal subunits and Nrf2 in the brain of DJ-1-deficient mice
To determine whether the proteasome activities are decreased due to lower levels of proteasome proteins, we examined selected subunits that are known to be important in overall proteasome activity. Low levels of 19S and 20S subunits of proteasome are found in PD patients (31,32) and this pathology has been suggested to contribute to proteasome dysfunction. 19S ATPase Rpt6 is a regulatory subunit, whose oxidative modification decreases the capability of proteasome to remove ubiquitinated proteins (43). The 20S ß5 subunit of the proteasome is responsible for chymotrypsin-like activity, which is a rate limiting activity of proteasome (44,45) and its expression is regulated by Nrf2 (46). Thus, experiments were carried out to investigate whether paraquat affects the protein levels of proteasome subunits in mouse brain (Fig. 6). Lysates of the striatum and VMB of DJ-1-deficient and wild-type mice treated with saline or paraquat were run on the gradient (4–20%) Tris–HCl SDS gels and protein levels of 19S ATPase Rpt6, 20S ß5 and Nrf2 were detected by Western blot analyses. There were significant effects of paraquat treatment on protein levels of 19S ATPase Rpt6, 20S ß5 and Nrf2. In addition, there were significant interactions of paraquat treatment and genotype for 20S ß5 subunit levels (P < 0.05 by two-way ANOVA). 19S ATPase Rpt6, 20S ß5 and Nrf2 protein levels in paraquat-treated DJ-1-deficient mice were significantly decreased to 65.4 ± 4.7% (P < 0.05, Nrf2), 70.6 ± 4.5% (P < 0.05, 20S ß5), and 53.9 ± 6.4% (P < 0.05, 19S ATPase Rpt6) of those in saline-treated DJ-1-deficient mice in VMB (Fig. 6F–H). Neither paraquat treatment nor DJ-1 deficiency had a significant effect on protein levels of these subunits and Nrf2 in the striatum (Fig. 6C–E).


Figure 6
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 6. Continued

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Our data demonstrate that paraquat produces abnormalities in motor behaviors and loss of dopamine in DJ-1-deficient mice whereas neither paraquat nor DJ-I deficiency alone does. This is accompanied by an increase in ubiquitinated proteins and a decrease in proteasome activities in the VMB. The reduction in proteasome activities is associated with a decrease in the regulatory subunit levels of 19S ATPase Rpt6 and 20S ß5 and as well as depletion of ATP, which is necessary for proteasome activity. Interestingly, we also note a decrease in Nrf2 levels. Nrf2 is a transcription factor that induces cytoprotective genes in response to various stressors (47) and is stabilized by DJ-1 (48). In addition, Nrf2 regulates an important proteasome subunit, 20S ß5 (46).

Our results demonstrate that the effect of paraquat is evident under the condition of DJ-1 deficiency in mice. This combined effect might represent activation of DJ-1 antioxidant function to alleviate oxidative stress produced by paraquat. In the presence of DJ-1, the effect of paraquat may be counteracted by DJ-1. Although dopamine levels were also decreased in paraquat-treated DJ-1-deficient mice, there was no significant cell loss consistent with a previous report on paraquat treatment of DJ-1-deficient mice (49). It is interesting that dopamine levels in DJ-1-deficient mice after paraquat treatment are similar to that of wild-type mice although behavioral deficit is present in the former group compared to the latter. This finding raises an important point that simple dopamine levels do not necessarily correlate linearly with final behavioral outcome, but the role of dopamine transmission and regulation in combination with other intracellular effects of DJ-1 deficiency may be responsible for the behavioral consequence. In addition, we cannot rule out a possibility that dysfunction in other brain areas may also contribute to overall behavioral abnormality.

Therefore, we focused our experiments on the effect of this combination of environmental and genetic factors on proteasome dysfunction, which is one of the key proposed mechanisms of pathology shared by DJ-1 and paraquat, as a potential explanation for the dopaminergic dysfunction that we observed. There is extensive evidence that proteasome impairment is associated with neurodegenerative diseases such as PD, Alzheimer disease, Huntington’s disease and prion disease (50,51). In particular, the decreases of proteasome activities and proteasome subunits and the formation of protein inclusions have been reported in PD brains (52). However, the mechanism of proteasome impairment in PD brains is not known.

The present study shows that paraquat in combination with DJ-1 deficiency decreases proteasome activities and increases the levels of ubiquitinated proteins in DJ-1-deficient mice (Figs 3 and 4). In particular, these phenomena were found in the VMB including SNpc, where similar pathologies have been reported in PD brains (30,52). Proteasome activity can be influenced by mitochondrial dysfunction, oxidative modification of proteasome subunits and the alteration of proteasome subunit levels (32,42,52). The impairment of proteasome function leads to the accumulation of ubiquitinated proteins and the formation of intracellular protein aggregates in vitro or in vivo (39,53). Paraquat can induce oxidative stress and mitochondrial dysfunction in brain tissues or cultured cells (10,54) and, therefore, could decrease proteasome activity accompanied by the aberrant accumulation of ubiquitinated proteins via such mechanisms. The antioxidant activity of DJ-1 has been tested in various model systems (55–57) and the deficiency of DJ-1 increases susceptibility to oxidative stress and produces mitochondrial dysfunction in cultured cells (23,58,59). Thus, our data suggest that two pathological conditions, exposure to paraquat and deficiency of DJ-1 might synergistically induce proteasome dysfunction accompanied by abnormal protein accumulation in the brain.

The current study demonstrates that paraquat decreased ATP levels in DJ-1-deficient mice (Fig. 5). This pathology was specifically observed in the VMB and not in the striatum. Mitochondrial complexes are tightly linked together to transport electron and produce ATP and thus, dysfunction of mitochondrial complexes might result in the inhibition of ATP production (39). In support of this idea, the inhibition of mitochondrial complex I leads to the decrease of ATP (60,61). Importantly, 19S ATPase subunit of the proteasome complex consumes ATP as an energy source for its function (40). Therefore, ATP depletion has been proposed to contribute to proteasome dysfunction (42) and this hypothesis is supported by an in vitro study (41). Paraquat induces mitochondrial dysfunction and ATP depletion in brain tissues or cultured cells (54,62). Therefore, our in vivo findings of ATP depletion would be a contributory factor for the observed proteasome dysfunction. DJ-1 has been suggested to contribute to mitochondrial integrity because DJ-1 is localized to mitochondrial matrix and inter-membrane space (63) and the oxidized form of DJ-1 moves into mitochondria, in response to paraquat-induced oxidative stress (64). Likewise, the knockdown of DJ-1 decreases the activity of mitochondrial complex I in cultured cells (58). Thus, our data suggest that two pathological conditions, exposure to paraquat and deficiency of DJ-1 might cooperatively induce ATP depletion and contribute to proteasome dysfunction in the brain.

The present study shows that paraquat decreases protein levels of proteasome subunits as well. Both 19S ATPase Rpt6 and 20S ß5 subunits and a transcription factor Nrf 2 were decreased in the VMB of DJ-1-deficient mice treated with paraquat (Fig. 6). The reduction of proteasome subunits is also reported in the same region of PD brains (31,32) and this defect has been suggested to contribute to proteasome dysfunction (9). In support of this hypothesis, some toxic agents reduce proteasome subunits in cultured cells or brain tissues (65,66). It is not known how DJ-1 affects the protein levels of proteasome subunits. However, two recent studies suggest a mechanism by which DJ-1 might be involved in the regulation of proteasome function. In particular, DJ-1 stabilizes a transcription factor Nrf2 (48), which regulates the expression of 20S ß5 subunit of proteasome (46). Thus, our data suggest that two pathological conditions, exposure to paraquat and deficiency of DJ-1 might jointly decrease protein levels of proteasome subunits, and contribute to proteasome dysfunction in the VMB. Therefore, the decrease in proteasomal activity is due to multiple factors including decrease in subunit protein levels as well as other factors such as ATP depletion.

In summary, the current findings provide the evidence that a combination of an environmental factor, paraquat and a genetic factor, DJ-1, might produce motor dysfunction, dopamine deficit and proteasome impairment in the mouse VMB. Future studies are necessary to elucidate the detailed mechanisms and causal relationships about how these two risk factors in combination contribute to these pathologies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
FUNDING
 REFERENCES
 
Materials
Suc-Leu-Leu-Val-Tyr-AMC and Z-Ala-Arg-Arg-AMC were purchased from Calbiochem (Calbiochem, San Diego, CA, USA). Z-Leu-Leu-Glu-AMC and a mouse monoclonal antibody to ß-actin antibody were purchased from Sigma (Sigma, St Louis, MO, USA). A rabbit polyclonal antibody to 20S ß5 and a mouse monoclonal antibody to 19S ATPase Rpt6 were purchased from Biomol (Biomol International LP, Plymouth, PA, USA). A rabbit polyclonal antibody to nuclear factor erythroid 2-related factor (Nrf2), a goat anti-rabbit secondary antibody and HeLa cell and THP-1 nuclear extract were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA). A rabbit polyclonal antibody to ubiquitinated protein was purchased from DAKO (DAKO, Fort Collins, CO, USA). ECL (enhanced chemiluminescence) Western blotting detection reagent was purchased from GE (GE Healthcare Life Sciences, Piscataway, NJ, USA). ENLIGHTEN ATP assay system bioluminescence detection kit and a goat anti-mouse secondary antibody were purchased from Promega (Promega, Madison, WI, USA). Gradient (4–20%) (w/v) sodium dodecyl sulfate polyacrylamide gels were purchased from Bio-rad (Bio-rad Laboratories, Hercules, CA, USA). Polyvinylidene fluoride (PVDF) membrane was purchased from Millipore Corporation (Millipore Corporation, Billerica, MA, USA). Bicinconinic acid (BCA) protein assay kit was purchased from Pierce (Pierce biotechnology, Rockford, IL, USA). The clear or white polystyrene 96-well plates were purchased from Fischer (Fischer Scientific Co., Pittsburgh, PA, USA). Unless indicated, all other chemicals were purchased from Sigma (Sigma, St Louis, MO, USA).

Animals, drug treatment and dissection of brain tissues
DJ-1-deficient mice previously generated were maintained in a climate and lighted room in the University of Chicago and genotyped as previously described (33). The DJ-1-deficient mice were originated in ES cells of 129 background, and bred to C57BL/6J wild-type, resulting in mixed 129xC57BL/6J background. Littermate wild-type mice were used as controls. For biochemical analyses, 4 to 6 month old male mice received intra-peritoneal injection with saline or 10 mg/kg paraquat dichloride once a week for 3 consecutive weeks. Then, mice were sacrificed by CO2 suffocation 2 days after last injection, and striatum and midbrain were immediately sliced at the thickness of 0.5 mm coronally in a mouse matrix (Zivic Laboratories Inc., Pittsburgh, PA, USA). Then, striatum was dissected at the coordinate between Bregma 1.34 and 0.38 mm (67), while the VMB mainly including substantia nigra area was obtained from the coronal slices (68). The dissected brain tissues were quickly frozen on dry ice and stored at –80°C until further processing. For behavioral and histological analyses, 4 to 6 month old male and female mice were tested 1 month after the last injection of paraquat and sacrificed for histological analysis after a couple of weeks of behavioral measurement. All animal procedures were approved by the Institutional Animal Care and Usage Committee of The University of Chicago.

Preparation of brain lysates
Frozen brain tissues were disrupted in a cold lysis buffer (10 mM HEPES, 10 mM Tris–HCl, pH 7.6, 0.5 mM EDTA, 1 mM dithiothreitol, 5 mM MgCl2) with a glass homogenizer followed by vigorous vortex for 60 s. Then, the homogenates were centrifuged at 15 000 g for 20 min at 4°C to remove cell debris and insoluble materials. The supernatants were collected and stored at –20°C for biochemical analyses. Protein concentrations of supernatants were determined using bicinconinc acid (BCA)-based protein assay kit.

Measurement of proteasome activities
Proteasome activities were measured as previously described with a slight modification (41,65,69). Lysates (50 µg) of the striatum and VMB were mixed with assay buffer (10 mM HEPES, 10 mM Tris–HCl, pH 7.6, 5 mM MgCl2, 1 mM dithiothreitol) in a clear polystyrene 96-well plate and then 100 µM fluorogenic substrates linked to 7-amido-4-methylcoumarin (AMC) were added to the mixture. In particular, Suc-Leu-Leu-Val-Tyr-AMC, Z-Ala-Arg-Arg-AMC and Z-Leu-Leu-Glu-AMC were used to measure chymotrypsin-like, trypsin-like or peptidylglutamyl-peptide hydrolyzing (PGPH)-like activity of proteasome, respectively. The final volume of mixtures was adjusted to 200 µl of final volume with assay buffer. After incubating the mixtures at 37°C for 30 min, the fluorescence signal released from free 7-amido-4-methylcoumarin (AMC) was detected at an excitation wavelength of 380 nm and an emission wavelength of 460 nm with a Tecan Safire2TM microplate reader (Tecan, Durham, NC, USA). The specific proteasome activity was determined as total activity minus remaining activity of lysates in the presence of 50 µM lactacystin.

Measurement of ATP levels
ATP levels were measured with an ATP assay kit, which utilizes luciferase to catalyze the formation of luminescence from ATP and luciferin as previously described with the following modification (70). Lysates (100 µg) of the striatum and VMB were mixed with 100 µl reconstituted solution of ATP assay kit in a white polystyrene 96-well plate. The final volume of mixtures was adjusted into 120 µl with a buffer (10 mM HEPES, 10 mM Tris-HCl, pH 7.6, 0.5 mM EDTA, 1 mM dithiothreitol, 5 mM MgCl2) used to prepare lysates. The luminescence was immediately measured with Tecan Safire2TM microplate reader (Tecan, Durham, NC, USA).

Western blotting
Lysates of the striatum and VMB (50 µg) were run on a gradient (4-20%) (w/v) sodium dodecyl sulfate polyacrylamide gel at 90 V for 2 h. After electrophoresis, the separated proteins were transferred at 130 mA, 50 V for 1 h onto a PVDF membrane. The membrane was blocked with 2.5% (w/v) non-fat dry milk solution in Tris-buffered saline-0.05% (v/v) Tween 20 (TBS-T) for 30 min and then incubated with primary antibodies (1:1000) at 4°C for 12 h. After incubation, the membrane was washed three times with Tris-buffered saline--0.05% (v/v) Tween 20 (TBS-T) at room temperature for 5 min and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000) for 3 h at room temperature. The membrane was washed three times with TBS-T at room temperature for 5 min. Protein bands were developed with ECL Western blotting detection reagents and then were visualized ChemiGenius Bio-Imaging system (Syngene, Frederick, MD, USA). The membranes were reprobed with ß-actin antibody (1:2500) to confirm the loading of equal protein amount. ImageJ version 1.33 (NIH, Maryland, MA, USA) was used to quantify each band area detected in blots.

Behavioral tests
A total of 32 males and females mice were used for behavioral tests. For Rotarod test, mice were first trained to stay on Rotarod (Columbus instrument) at a constant speed of 5 rpm for at least 1 min. Following training, mice were tested for a total of three trials with an accelerating speed of 0.2 rpm/s, starting at 5 rpm. The latency to fall was recorded for each trial and the average of three trials was reported. For open field test, each mouse was placed in an open field chamber (40 cm long x 40 cm wide x 37 cm high, Med Associates) and habituated for 10 min. Illumination of open filed was set to 20 lux. No background noise was provided. They were monitored by infrared beams that record the animal’s location and path (locomotor activity) as well as the number of rearing movements (vertical activity). Data were collected for 30 min.

Stereological dopamine neuron counting and HPLC
After behavioral tests, mice were perfused with PBS. Brains were cut into halves along the midline. Striata and VMBs of right half brains were dissected on ice, frozen in dry ice powder and stored in –80°C for HLPC. Left half brains were post-fixed in 4% (w/v) paraformaldehyde at 4°C for 1 week. The fixed brains were cryoprotected in 30% (w/v) sucrose. The 40 µm sections were cut on. Stereological dopamine neuron counting was performed on 32 mice used in behavioral tests and HPLC was performed in a total of 64 mice. The procedures of stereological dopamine neuron counting and HPLC were described in our previous publication (33).

Statistical analysis
All values are presented as mean ± SEM. Statistical significance was determined with two-way analysis of variance (ANOVA) and the Bonferronìs test for multiple comparison test (SigmaStat Statistical Software version 2.03). A P-value less than 0.05 was considered to be significant.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
REFERENCES
 
This project is supported by grants from National Institute of Health (NS053919 to UK, NS043286 to UK), American Parkinson Disease Association Center for Advanced Research (to UK) and an International Grant from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, the Republic of Korea (to UK).

Conflict of Interest statement. None declared.


   ACKNOWLEDGEMENTS
 
We thank William Lin and Lisa Won for their helpful discussions and comments on the manuscript and Wanhao Chi for her expert technical help.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 

  1. de Rijk M.C., Breteler M.M., Graveland G.A., Ott A., Grobbee D.E., van der Meche F.G., Hofman A. Prevalence of Parkinson’s disease in the elderly: The Rotterdam Study. Neurology (1995) 45:2143–2146.[Abstract/Free Full Text]

  2. Wood-Kaczmar A., Gandhi S., Wood N.W. Understanding the molecular causes of Parkinson’s disease. Trends Mol. Med. (2006) 12:521–528.[CrossRef][Web of Science][Medline]

  3. Lang A.E., Lozano A.M. Parkinson’s disease. Second of two parts. N. Engl. J. Med. (1998) 339:1130–1143.[Free Full Text]

  4. Forno L.S. Neuropathology of Parkinson’s disease. J. Neuropathol. Exp. Neurol. (1996) 55:259–272.[Web of Science][Medline]

  5. Hertzman C., Wiens M., Bowering D., Snow B., Calne D. Parkinson‘s disease: a case-control study of occupational and environmental risk factors. Am. J. Ind. Med. (1990) 17:349–355.[Web of Science][Medline]

  6. Liou H.H., Tsai M.C., Chen C.J., Jeng J.S., Chang Y.C., Chen S.Y., Chen R.C. Environmental risk factors and Parkinson‘s disease: a case-control study in Taiwan. Neurology (1997) 48:1583–1588.[Abstract]

  7. Sanchez-Ramos J.R., Hefti F., Weiner W.J. Paraquat and Parkinson‘s disease. Neurology (1987) 37:728.[Free Full Text]

  8. Liou H.H., Chen R.C., Tsai Y.F., Chen W.P., Chang Y.C., Tsai M.C. Effects of paraquat on the substantia nigra of the Wistar rats: neurochemical, histological, and behavioral studies. Toxicol. Appl. Pharmacol. (1996) 137:34–41.[CrossRef][Web of Science][Medline]

  9. McCormack A.L., Thiruchelvam M., Manning-Bog A.B., Thiffault C., Langston J.W., Cory-Slechta D., Di Monte D.A. Environmental risk factors and Parkinson‘s disease: selective degeneration of nigral dopaminergic neurons caused by the herbicide paraquat. Neurobiol. Dis. (2002) 19:119–127.

  10. McCormack A.L., Atienza J.G., Johnston L.C., Andersen J.K., Vu S., Di Monte D.A. Role of oxidative stress in paraquat-induced dopaminergic cell degeneration. J. Neurochem. (2005) 93:1030–1037.[CrossRef][Web of Science][Medline]

  11. Manning-Bog A.B., McCormack A.L., Li J., Uversky V.N., Fink A.L., Di Monte D.A. The herbicide paraquat causes up-regulation and aggregation of {alpha}-synuclein in mice. J. Biol. Chem. (2002) 277:1641–1644.[Abstract/Free Full Text]

  12. Tao X., Tong L. Crystal structure of human DJ-1, a protein associated with early onset Parkinson’s disease. J. Biol. Chem. (2003) 278:31372–31379.[Abstract/Free Full Text]

  13. Bonifati V., Oostra B.A., Heutink P. Linking DJ-1 to neurodegeneration offers novel insights for understanding the pathogenesis of Parkinson’s disease. J. Mol. Med. (2004) 82:163–174.[CrossRef][Web of Science][Medline]

  14. Bandopadhyay R., Kingsbury A.E., Cookson M.R., Reid A.R., Evans I.M., Hope A.D., Pittman A.M., Lashley T., Canet-Aviles R., Miller D.W., et al. The expression of DJ-1 (PARK7) in normal human CNS and idiopathic Parkinson’s disease. Brain (2004) 127:420–430.[Abstract/Free Full Text]

  15. Nagakubo D., Taira T., Kitaura H., Ikeda M., Tamai K., Iguchi-Ariga S.M., Ariga H. DJ-1, a novel oncogene which transforms mouse NIH3T3 cells in cooperation with ras. Biochem. Biophys. Res. Commun. (1997) 231:509–513.[CrossRef][Web of Science][Medline]

  16. Taira T., Saito Y., Niki T., Iguchi-Ariga S.M., Takahashi K., Ariga H. DJ-1 has a role in antioxidative stress to prevent cell death. EMBO Rep. (2004) 5:213–218.[CrossRef][Web of Science][Medline]

  17. Takahashi K., Taira T., Niki T., Seino C., Iguchi-Ariga S.M., Ariga H. DJ-1 positively regulates the androgen receptor by impairing the binding of PIASx alpha to the receptor. J. Biol. Chem. (2001) 276:37556–37563.[Abstract/Free Full Text]

  18. Xu J., Zhong N., Wang H., Elias J.E., Kim C.Y., Woldman I., Pifl C., Gygi S.P., Geula C., Yankner B.A. The Parkinson’s disease-associated DJ-1 protein is a transcriptional co-activator that protects against neuronal apoptosis. Hum. Mol. Genet. (2005) 14:1231–1241.[Abstract/Free Full Text]

  19. Zhong N., Kim C.Y., Rizzu P., Geula C., Porter D.R., Pothos E.N., Squitieri F., Heutink P., Xu J. DJ-1 transcriptionally up-regulates the human tyrosine hydroxylase by inhibiting the sumoylation of pyrimidine tract-binding protein-associated splicing factor. J. Biol. Chem. (2006) 281:20940–20948.[Abstract/Free Full Text]

  20. Martinat C., Shendelman S., Jonason A., Leete T., Beal M.F., Yang L., Floss T., Abeliovich A. Sensitivity to oxidative stress in DJ-1-deficient dopamine neurons: an ES- derived cell model of primary Parkinsonism. PLoS Biol. (2004) 2:e327.[CrossRef][Medline]

  21. Yokota T., Sugawara K., Ito K., Takahashi R., Ariga H., Mizusawa H. Down regulation of DJ-1 enhances cell death by oxidative stress, ER stress, and proteasome inhibition. Biochem. Biophys. Res. Commun. (2003) 312:1342–1348.[CrossRef][Web of Science][Medline]

  22. Shendelman S., Jonason A., Martinat C., Leete T., Abeliovich A. DJ-1 is a redox-dependent molecular chaperone that inhibits alpha-synuclein aggregate formation. PLoS Biol. (2004) 2:e362.[CrossRef][Medline]

  23. Zhou W., Freed C.R. DJ-1 up-regulates glutathione synthesis during oxidative stress and inhibits A53T alpha-synuclein toxicity. J. Biol. Chem. (2005) 280:43150–43158.[Abstract/Free Full Text]

  24. DeMartino G.N., Slaughter C.A. The proteasome, a novel protease regulated by multiple mechanisms. J. Biol. Chem. (1999) 274:22123–22126.[Free Full Text]

  25. Tanaka K., Chiba T. The proteasome: a protein-destroying machine. Genes Cells (1998) 3:499–510.[Abstract]

  26. de Vrij F.M., Fischer D.F., van Leeuwen F.W., Hol E.M. Protein quality control in Alzheimer’s disease by the ubiquitin proteasome system. Prog. Neurobiol. (2004) 74:249–270.[CrossRef][Web of Science][Medline]

  27. Goldberg A.L. Protein degradation and protection against misfolded or damaged proteins. Nature (2003) 426:895–899.[CrossRef][Medline]

  28. Sherman N.Y., Goldberg A.L. Cellular defenses against unfolded proteins: a cell biologist thinks about neurodegenerative diseases. Neuron (2001) 29:15–32.[CrossRef][Web of Science][Medline]

  29. Chung K.K., Dawson V.L., Dawson T.M. The role of the ubiquitin-proteasomal pathway in Parkinson’s disease and other neurodegenerative disorders. Trends Neurosci. Suppl. (2001) 24:S7–S14.[CrossRef]

  30. McNaught K.S., Jenner P. Proteasomal function is impaired in substantia nigra in Parkinson’s disease. Neurosci. Lett. (2001) 297:191–194.[CrossRef][Web of Science][Medline]

  31. McNaught K.S., Belizaire R., Jenner P., Olanow C.W., Isacson O. Selective loss of 20S proteasome alpha-subunits in the substantia nigra pars compacta in Parkinson’s disease. Neurosci. Lett. (2002) 326:155–158.[CrossRef][Web of Science][Medline]

  32. McNaught K.S., Belizaire R., Isacson O., Jenner P., Olanow C.W. Altered proteasomal function in sporadic Parkinson’s disease. Exp. Neurol. (2003) 179:38–46.[CrossRef][Web of Science][Medline]

  33. Chen L., Cagniard B., Mathews T., Jones S., Koh H.C., Ding Y., Carvey P.M., Ling Z., Kang U.J., Zhuang X. Age-dependent motor deficits and dopaminergic dysfunction in DJ-1 null mice. J. Biol. Chem. (2005) 280:21418–21426.[Abstract/Free Full Text]

  34. Brooks A.I., Chadwick H.A., Gelbard D.A., Cory-Slechta D.A., Federoff H.J. Paraquat elicited neurobehavioral syndrome caused by dopaminergic neuron loss. Brain Res. (1999) 823:1–10.[CrossRef][Web of Science][Medline]

  35. Thiruchelvam M., Richfield E.K., Baggs R.B., Tank A.W., Cory-Slechta D.A. The nigrostriatal dopaminergic system as a preferential target of repeated exposures to combined paraquat and maneb: implications for Parkinson’s disease. J. Neurosci. (2000) 20:9207–9214.[Abstract/Free Full Text]

  36. Thiruchelvam M., Prokopenko O., Cory-Slechta D.A., Richfield E.K., Buckley B., Mirochnitchenko O. Overexpression of superoxide dismutase or glutathione peroxidase protects against the paraquat + maneb-induced Parkinson disease phenotype. J. Biol. Chem. (2005) 280:22530–22539.[Abstract/Free Full Text]

  37. Dick T.P., Nussbaum A.K., Deeg M., Heinemeyer W., Groll M., Schirle M., Keilholz W., Stevanovic S., Wolf D.H., Huber R., et al. Contribution of proteasomal beta-subunits to the cleavage of peptide substrates analyzed with yeast mutants. J. Biol. Chem. (1998) 273:25637–25646.[Abstract/Free Full Text]

  38. Hyun D.H., Gray D.A., Halliwell B., Jenner P. Interference with ubiquitination causes oxidative damage and increased protein nitration: implications for neurodegenerative diseases. J. Neurochem. (2004) 90:422–430.[CrossRef][Web of Science][Medline]

  39. Rideout H.J., Stefanis L. Proteasomal inhibition-induced inclusion formation and death in cortical neurons require transcription and ubiquitination. Mol. Cell Neurosci. (2002) 21:223–238.[CrossRef][Web of Science][Medline]

  40. Smith D.M., Benaroudj N., Goldberg A. Proteasomes and their associated ATPases: a destructive combination. J. Struct. Biol. (2006) 156:72–83.[Web of Science][Medline]

  41. Höglinger G.U., Carrard G., Michel P.P., Medja F., Lombès A., Ruberg M., Friguet B., Hirsch E.C. Dysfunction of mitochondrial complex I and the proteasome: interactions between two biochemical deficits in a cellular model of Parkinson’s disease. J. Neurochem. (2003) 86:1297–1307.[Web of Science][Medline]

  42. McNaught K.S., Jackson T., JnoBaptiste R., Kapustin A., Olanow C.W. Proteasomal dysfunction in sporadic Parkinson‘s disease. Neurology (2006) 66:S37–S49.[Abstract/Free Full Text]

  43. Ishii T., Sakurai T., Usami H., Uchida K. Oxidative modification of proteasome: identification of an oxidation-sensitive subunit in 26 S proteasome. Biochemistry (2005) 44:13893–13901.[CrossRef][Medline]

  44. Groll M., Heinemeyer W., Jäger S., Ullrich T., Bochtler M., Wolf D.H., Huber R. The catalytic sites of 20S proteasomes and their role in subunit maturation: a mutational and crystallographic study. Proc. Natl Acad. Sci. USA (1999) 96:10976–10983.[Abstract/Free Full Text]

  45. Li Z., Arnaud L., Rockwell P., Figueiredo-Pereira M.E. A single amino acid substitution in a proteasome subunit triggers aggregation of ubiquitinated proteins in stressed neuronal cells. J. Neurochem. (2004) 90:19–28.[CrossRef][Medline]

  46. Kwak M.K., Kensler T.W. Induction of 26S proteasome subunit PSMB5 by the bifunctional inducer 3-methylcholanthrene through the Nrf2-ARE, but not the AhR/Arnt-XRE, pathway. Biochem. Biophys. Res. Commun. (2006) 345:1350–1357.[CrossRef][Web of Science][Medline]

  47. Kensler T.W., Wakabayashi N., Biswal S. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu. Rev. Pharmacol. Toxicol. (2007) 47:89–116.[CrossRef][Web of Science][Medline]

  48. Clements C.M., McNally R.S., Conti B.J., Mak T.W., Ting J.P. DJ-1, a cancer- and Parkinson’s disease-associated protein, stabilizes the antioxidant transcriptional master regulator Nrf2. Proc. Natl Acad. Sci. USA (2006) 103:15091–15096.[Abstract/Free Full Text]

  49. Goldberg M.S., Pisani A., Haburcak M., Vortherms T.A., Kitada T., Costa C., Tong Y., Martella G., Tscherter A., Martins A., et al. Nigrostriatal dopaminergic deficits and hypokinesia caused by inactivation of the familial parkinsonism-linked gene DJ-1. Neuron (2005) 45:489–496.[CrossRef][Web of Science][Medline]

  50. Barnham K.J., Masters C.L., Bush A.I. Neurodegenerative diseases and oxidative stress. Nat. Rev. Drug Discov. (2004) 3:205–214.[CrossRef][Web of Science][Medline]

  51. Ciechanover A., Brundin P. The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg. Neuron (2003) 40:427–446.[CrossRef][Web of Science][Medline]

  52. McNaught K.S., Olanow C.W. Protein aggregation in the pathogenesis of familial and sporadic Parkinson’s disease. Neurobiol. Aging (2006) 27:530–545.[CrossRef][Web of Science][Medline]

  53. McNaught K.S., Perl D.P., Brownell A.L., Olanow C.W. Systemic exposure to proteasome inhibitors causes a progressive model of Parkinson’s disease. Ann. Neurol. (2004) 56:149–162.[CrossRef][Web of Science][Medline]

  54. Gomez C., Bandez M.J., Navarro A. Pesticides and impairment of mitochondrial function in relation with the parkinsonian syndrome. Front Biosci. (2007) 12:1079–1093.[CrossRef][Web of Science][Medline]

  55. Inden M., Taira T., Kitamura Y., Yanagida T., Tsuchiya D., Takata K., Yanagisawa D., Nishimura K., Taniguchi T., Kiso Y., et al. PARK7 DJ-1 protects against degeneration of nigral dopaminergic neurons in Parkinson’s disease rat model. Neurobiol. Dis. (2006) 24:144–158.[CrossRef][Web of Science][Medline]

  56. Li H.M., Taira T., Maita C., Ariga H., Iguchi-Ariga S.M. Protection against nonylphenol-induced cell death by DJ-1 in cultured Japanese medaka (Oryzias latipes) cells. Toxicology (2007) 228:229–238.[Web of Science]

  57. Menzies F.M., Yenisetti S.C., Min K.T. Roles of Drosophila DJ-1 in survival of dopaminergic neurons and oxidative stress. Curr. Biol. (2005) 15:1578–1582.[CrossRef][Web of Science][Medline]

  58. Ooe H., Taira T., Iguchi-Ariga S.M., Ariga H. Induction of reactive oxygen species by bisphenol A and abrogation of bisphenol A-induced cell injury by DJ-1. Toxicol. Sci (2005) 88:114–126.[Abstract/Free Full Text]

  59. Yang Y., Gehrke S., Haque M.E., Imai Y., Kosek J., Yang L., Beal M.F., Nishimura I., Wakamatsu K., Ito S., et al. Inactivation of Drosophila DJ-1 leads to impairments of oxidative stress response and phosphatidylinositol 3-kinase/Akt signaling. Proc. Natl Acad. Sci. USA (2005) 102:13670–13675.[Abstract/Free Full Text]

  60. Hirata Y., Nagatsu T. Rotenone and CCCP inhibit tyrosine hydroxylation in rat striatal tissue slices. Toxicology (2005) 216:9–14.[CrossRef][Web of Science][Medline]

  61. Sherer T.B., Betarbet R., Testa C.M., Seo B.B., Richardson J.R., Kim J.H., Miller G.W., Yagi T., Matsuno-Yagi A., Greenamyre J.T. Mechanism of toxicity in rotenone models of Parkinson’s disease. J. Neurosci. (2003) 23:10756–10764.[Abstract/Free Full Text]

  62. Maracchioni A., Totaro A., Angelini D.F., Di Penta A., Bernardi G., Carrì M.T., Achsel T. Mitochondrial damage modulates alternative splicing in neuronal cells: implications for neurodegeneration. J. Neurochem. (2007) 100:142–153.[CrossRef][Web of Science][Medline]

  63. Zhang L., Shimoji M., Thomas B., Moore D.J., Yu S.W., Marupudi N.I., Torp R., Torgner I.A., Ottersen O.P., Dawson T.M., et al. Mitochondrial localization of the Parkinson’s disease related protein DJ-1: implications for pathogenesis. Hum. Mol. Genet. (2005) 14:2063–2073.[Abstract/Free Full Text]

  64. Canet-Avilés R.M., Wilson M.A., Miller D.W., Ahmad R., McLendon C., Bandyopadhyay S., Baptista M.J., Ringe D., Petsko G.A., Cookson M.R. The Parkinson’s disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization. Proc. Natl Acad. Sci. USA (2004) 101:9103–9108.[Abstract/Free Full Text]

  65. Betarbet R., Canet-Aviles R.M., Sherer T.B., Mastroberardino P.G., McLendon C., Kim J.H., Lund S., Na H.M., Taylor G., Bence N.F., et al. Intersecting pathways to neurodegeneration in Parkinson’s disease: effects of the pesticide rotenone on DJ-1, alpha-synuclein, and the ubiquitin-proteasome system. Neurobiol. Dis. (2006) 22:404–420.[CrossRef][Web of Science][Medline]

  66. Shamoto-Nagai M., Maruyama W., Akao Y., Osawa T., Tribl F., Gerlach M., Zucca F.A., Zecca L., Riederer P., Naoi M. Neuromelanin inhibits enzymatic activity of 26S proteasome in human dopaminergic SH-SY5Y cells. J. Neural. Transm. (2004) 111:1253–1265.[CrossRef][Web of Science][Medline]

  67. Franklin K.B., Paxinos G. The Mouse Brain in Stereotaxic Coordinates. (1997) San Diego, USA: Academic Press.

  68. Ding Y.M., Jaumotte J.D., Signore A.P., Zigmond M.J. Effects of 6-hydroxydopamine on primary cultures of substantia nigra: specific damage to dopamine neurons and the impact of glial cell line-derived neurotrophic factor. J. Neurochem. (2004) 89:776–787.[CrossRef][Web of Science][Medline]

  69. Zeng B.Y., Iravani M.M., Lin S.T., Irifune M., Kuoppamäki M., Al-Barghouthy G., Smith L., Jackson M.J., Rose S., Medhurst A.D., et al. MPTP treatment of common marmosets impairs proteasomal enzyme activity and decreases expression of structural and regulatory elements of the 26S proteasome. Eur. J. Neurosci. (2006) 23:1766–1774.[CrossRef][Web of Science][Medline]

  70. Abrahams M.R., Zhang Z., Chien S., Skerns T., Kotwal G.J. The vaccinia virus N1L ORF may encode a multifunctional protein possibly targeting different kinases, one of which influences ATP levels in vivo. Ann. N. Y. Acad. Sci. (2005) 1056:87–99.[CrossRef][Web of Science][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
16/23/2900    most recent
ddm249v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Yang, W.
Right arrow Articles by Kang, U. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, W.
Right arrow Articles by Kang, U. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?