ToxSci Advance Access originally published online on October 12, 2006
Toxicological Sciences 2007 95(1):196-204; doi:10.1093/toxsci/kfl133
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Obligatory Role for Complex I Inhibition in the Dopaminergic Neurotoxicity of 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
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* Department of Environmental and Occupational Medicine, University of Medicine and Dentistry–New Jersey/Robert Wood Johnson Medical School and Environmental and Occupational Health Sciences Institute, Piscataway, New Jersey 08854
Center for Neurodegenerative Disease, School of Medicine, Emory University, Atlanta, Georgia 30322
Department of Environmental and Occupational Health, Rollins School of Public Health, Emory University, Atlanta, Georgia 30322
Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037
¶ Department of Neurology and Pittsburgh Institute for Neurodegenerative Diseases, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
2 To whom correspondence should be addressed at Center for Neurodegenerative Disease, School of Medicine, Emory University, Whitehead Biomedical Research Building Room 505, 615 Michael Street, Atlanta, GA 30322. Fax: (404) 727-3728. E-mail: gary.miller{at}emory.edu.
Received August 25, 2006; accepted October 7, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mice and nonhuman primates causes a parkinsonian disorder characterized by a loss of dopamine-producing neurons in the substantia nigra and corresponding motor deficits. MPTP has been proposed to exert its neurotoxic effects through a variety of mechanisms, including inhibition of complex I of the mitochondrial respiratory chain, displacement of dopamine from vesicular stores, and formation of reactive oxygen species from mitochondrial or cytosolic sources. However, the mechanism of MPTP-induced neurotoxicity is still a matter of debate. Recently, we reported that the yeast single-subunit nicotinamide adenine dinucleotide (reduced) dehydrogenase (NDI1) is resistant to rotenone, a complex I inhibitor that produces a parkinsonian syndrome in rats, and that overexpression of NDI1 in SK-N-MC cells prevents the toxicity of rotenone. In this study, we used viral-mediated overexpression of NDI1 in SK-N-MC cells and animals to determine the relative contribution of complex I inhibition in the toxicity of MPTP. In cell culture, NDI1 overexpression abolished the toxicity of 1-methyl-4-phenylpyridinium, the active metabolite of MPTP. Overexpression of NDI1 through stereotactic administration of a viral vector harboring the NDI1 gene into the substantia nigra protected mice from both the neurochemical and behavioral deficits elicited by MPTP. These data identify inhibition of complex I as a requirement for dopaminergic neurodegeneration and subsequent behavioral deficits produced by MPTP. Furthermore, combined with reports of a complex I defect in Parkinson's disease (PD) patients, the present study affirms the utility of MPTP in understanding the molecular mechanisms underlying dopaminergic neurodegeneration in PD.
Key Words: Mitochondria; complex I; MPTP; Parkinson's disease; dopamine transporter; viral expression.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Parkinson's disease (PD) is a progressive neurodegenerative disorder estimated to affect greater than 1% of all adults over the age of 65 worldwide (Olanow and Tatton, 1999
). Although genetic mutations can cause PD, the vast majority of cases are idiopathic (Vila and Przedborski, 2004) and there is increasing evidence that exposure to environmental toxicants may play a significant role in PD (Sherer et al., 2002). A key indication that environmental factors may play a role in PD came with the discovery of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which caused an acute and permanent parkinsonian syndrome in intravenous drug users (Langston et al., 1983). Since this discovery, MPTP has been used to develop animal models of PD in several species, as a tool to understand the pathogenesis of PD and to test a variety of therapeutic interventions designed to prevent or reduce dopaminergic neurodegeneration (Dauer and Prezdborski, 2003).
Despite intensive investigation, the molecular mechanism of MPTP has not been definitively demonstrated. MPP+, the active metabolite of MPTP, has been shown to inhibit complex I of the mitochondrial electron transport chain, which was thought to be the mechanism by which MPTP induced parkinsonism (Nicklas et al., 1985). However, the weak inhibitory ability of MPP+ at complex I (Ramsay et al., 1991; Richardson et al., 2005) has led some to question the role of complex I inhibition in MPTP toxicity (Lotharius and O'Malley, 2000; Nakamura et al., 2000). Unfortunately, complex I activity was not determined in these studies and the contribution of complex I in the toxicity of MPP+ could not directly be determined. Others have hypothesized alternate mechanisms of action for MPTP including release of vesicular stored dopamine and subsequent oxidative damage, release of stored iron deposits, increased cytoplasmic calcium and intraneuronal calcium release, and redox cycling of MPP+ (Chen et al., 1995; Kooncumchoo et al., 2006; Lotharius and O'Malley, 2000; Obata, 2002, 2006; Trevor et al., 1987). Thus, the molecular mechanism of MPTP remains to be established.
Because the administration of MPTP to experimental animals is the most widely used model of PD, determination of the mechanism by which MPTP causes dopaminergic neurodegeneration may provide critical information on pathogenic process involved in PD. Previously, we have demonstrated that the single-subunit nicotinamide adenine dinucleotide (reduced) dehydrogenase of Saccharomyces cerevisiae (NDI1) can serve as a replacement for complex I in mammalian cells and restore electron transfer in cell devoid of mitochondrial DNA (Bai et al., 2001; Seo et al., 1998, 2000) and that NDI1 expression in neuroblastoma cells abolishes the toxicity of rotenone, a complex I inhibitor we have demonstrated to reproduce features of PD in rats (Betarbet et al., 2000; Sherer et al., 2003). More recently, we have reported that NDI1 can be expressed in vivo using viral-mediated techniques and that unilateral expression of NDI1 partially protected against reduced tyrosine hydroxylase (TH) immunoreactivity resulting from an acute high-dose regimen of MPTP (4 x 15 mg/kg over 1 day; Seo et al., 2006). These findings suggested that NDI1 could provide some protection from MPTP neurotoxicity. However, the in vivo nature of our experiments prevented unequivocal identification of complex I as the target of MPTP. Additionally, the unilateral nature of our experiments did not allow us to determine whether NDI1-mediated neuroprotection would ameliorate the behavioral deficits observed following MPTP administration (Tillerson et al., 2002). Here, we sought to definitively determine the role of complex I inhibition in the neurotoxicity produced by MPTP. Using an in vitro system, we demonstrate that overexpression of NDI1 in cells totally abolishes the toxicity of MPP+, the active metabolite of MPTP. Furthermore, viral-mediated overexpression of NDI1 in vivo resulted in significant reduction of MPTP dopaminergic neurotoxicity and the total recovery of behavioral deficits caused by MPTP.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture.
SK-N-MC neuroblastoma cells stably expressing NDI1 protein have been described previously (Sherer et al., 2003
). To demonstrate proper localization of NDI1 to the mitochondria, immunofluorescence microscopy was performed as described previously (Seo et al., 2000). Briefly, cells were fixed and immunostained with a previously characterized affinity-purified rabbit antibody to NDI1 and an antibody to the mitochondrial F1-adenosine triphosphatase (ATPase) (Molecular Probes, Eugene, OR). Secondary antibodies were FITC-conjugated goat anti-rabbit for NDI1 and rhodamine red goat anti-mouse for F1-ATPase (Molecular Probes). For studies on MPP+ toxicity, parental SK-N-MC or SK-N-MC cells stably expressing NDI1 (Sherer et al., 2003) were transfected with 4 µg of the dopamine transporter (DAT) in pcDNA 3.1 using Lipofectamine as described previously (Ramachandiran et al., 2006). Twenty-four hours after transfection, cells were split into 24-well plates for determination of 1-methyl-4-phenylpyridinium (3H-MPP+) uptake (Pifl et al., 1993) on the following day (48 h after transfection). Briefly, cells were washed with 1 ml uptake buffer (4mM Tris, 6.25mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 120mM NaCl, 5mM KCl, 1.2mM CaCl2, 1.2mM MgSO4, 5.6mM D-glucose, and 0.5mM ascorbic acid, pH 7.2) and incubated in 0.5 ml uptake buffer containing various concentrations (0.5–40µM) of cold MPP+ and 0.25 µCi of 3H-MPP+ for 10 min at 37°C. Nonspecific uptake was determined in the presence of 10µM nomifensine. Uptake was terminated by aspirating the uptake buffer and washing each well twice with 1 ml of ice-cold uptake buffer. Cells were lysed by 1% sodium dodecyl sulfate and transferred into vials containing 5 ml of scintillation cocktail and radioactivity measured by liquid scintillation counting. Uptake values were corrected for the amount of protein to account for variation in plating density and kinetic (Km and Vmax) values were calculated by nonlinear regression using GraphPad Prism 4.0. For studies on cell death, SK-N-MC cells stably expressing NDI1 were transfected with the DAT as described above. Cells were then incubated for 48 h with 100µM MPP+ and cell death determined using Sytox green (Molecular Probes, Eugene, OR) with fluorescent detection as described previously (Sherer et al., 2003). Following the last measurement, cells were incubated for 1 h with 1% Triton, and total fluorescence was determined to normalize for variation in plating density.
Animal studies.
Eight-week-old male C57BL/6j mice were obtained from an in-house breeding colony at the Scripps Research Institute. Mice received stereotaxic injections (anterior-posterior –3.3 mm, medial-lateral + 1.4 mm, and dorso-ventral –3.9 mm) of phosphate-buffered saline vehicle (n = 14) or rAAV-NDI1 (n = 17), a recombinant adeno-associated serotype 2 virus carrying the NDI1 gene (1 x 1011 IU/ml in phosphate-buffered saline), bilaterally into the substantia nigra (Seo et al., 2004, 2006). A total of 31 mice were shipped to Emory University at 4–5 months of age and quarantined for 3 months. Therefore, mice were 8 months of age at the time of experimentation, an age at which MPTP produces a sustained loss of dopaminergic terminals and cell bodies (Ali et al., 1991; Tillerson et al., 2002). Mice were then assigned to one of four treatment groups: (1) vehicle-saline (n = 3); (2) vehicle-MPTP (2 x 15 mg/kg; n = 6); (3) NDI1-saline (n = 5); and (4) NDI1-MPTP (n = 12). The use of a higher number of animals in the NDI1-MPTP group was based on previous observations that the expression of NDI1 varies from animal to animal (Seo et al., 2006). Animals underwent behavioral testing 7 days following MPTP treatment and were then sacrificed (Tillerson and Miller, 2003). The brain was removed and divided at the midline, with one half of the brain placed in freshly prepared 4% paraformaldehyde and the striatum dissected from the other side and frozen for neurochemical determinations. All animal procedures were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and approved by both the Ethical Committee for Use of Laboratory Animals at the Scripps Research Institute and the Institutional Animal Care and Use Committee at Emory University.
Neurochemical determinations.
Western blots were performed as previously described (Richardson et al., 2006). Briefly, samples (20 µg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 10% precast NuPage gels (Invitrogen, Carlsbad, CA). Samples were electrophoretically transferred to a polyvinylidene difluoride membrane, and nonspecific sites were blocked in 7.5% nonfat dry milk in Tris-buffered saline (135mM NaCl, 2.5mM KCl, 50mM Tris, and 0.1% Tween 20, pH 7.4). Membranes were then incubated in a monoclonal antibody (Chemicon, Temecula, CA) to the N-terminus of DAT. Antibody binding was detected using a goat anti-rat horseradish peroxidase secondary antibody (ICN, Costa Mesa, CA) and enhanced chemiluminescence. The chemiluminescent signal was captured on an Alpha Innotech Fluorchem 8800 (San Leandro, CA) imaging system and stored as a digital image. Densitometric analysis was performed and calibrated to coblotted dilutional standards of pooled cells from all control samples. Membranes were then stripped for 15 min at 25°C with Pierce (Rockford, IL) Stripping Buffer and sequentially reprobed with polyclonal anti-TH, polyclonal anti-vesicular monoamine transporter 2 (VMAT2), and monoclonal anti-ß-actin (Sigma-Aldrich, St Louis, MO) antibody to ensure equal protein loading across samples. For assessment of astroglial and microglial activation, blots were probed with polyclonal antibodies to glial fibrillary acidic protein (GFAP) and glucose transporter-5 (Glut5).
High-performance liquid chromatography (HPLC) analysis of catecholamines by electrochemical detection was performed as previously described (Richardson and Miller, 2004). Briefly, dissected striata were sonicated in 0.1M perchloric acid containing 347µM sodium bisulfite and 134µM ethylenediaminetetraacetic acid. Homogenates were centrifuged at 12,000 x g for 10 min at 4°C, the supernatant was removed, and filtered through a 0.22-µm filter by centrifugation at 12,000 x g for 10 min. The supernatants were then analyzed for levels of dopamine, dihydroxyphenylacetic acid, and homovanillic acid. Levels were measured using HPLC with an eight-channel coulometric electrode array (ESA Coularray, Chelmsford, MA). Quantification was made by reference to calibration curves made with individual monoamine standards.
Immunohistochemistry.
Immunohistochemical analysis of tissue was performed as previously described by Caudle et al. (2006). Brains were serially sectioned at 25 µm on a freezing microtome. Tissue was rinsed in 1x TBS and incubated in 3% H2O2 to quench endogenous peroxidases. Sections were blocked in 10% normal goat serum for 1 h at room temperature before being incubated overnight with monoclonal mouse anti-TH (1:2000) primary antibody. The following day tissue was rinsed in 1x TBS then incubated for 1 h in goat anti-mouse (1:200) secondary antibody conjugated to biotin. Tissue was rinsed in 1x TBS and incubated for 1 h in avidin-biotin complex solution. Tissue was rinsed in 1x TBS and the final product was visualized using 3,3'-diaminobenzidine tetrachloride. Free-floating slices were mounted onto slides, serially dehydrated in ethanol, and coverslipped. Immunostained sections were analyzed using bright-field microscopy, and images were captured on a Leitz microscope (Leica, Wetzlar, Germany). For final output, images were processed simultaneously and identically with Adobe Photoshop 7.0 software.
Behavioral testing.
Behavioral testing to determine the behavioral impact of MPTP administration was performed via the grid performance test as described previously (Tillerson and Miller, 2003; Tillerson et al., 2002). Briefly, mice were lifted by their tail and slowly placed in the center of the horizontal grid and supported until they grabbed the grid with both their fore and hind-paws. The grid was then inverted and mice were videotaped while hanging upside down for 30 s. For analysis, videos were replayed using a recorder with slow motion and frame-by-frame option by a person blinded to the treatment groups. Forepaw step length was measured as the number of grid squares/openings transversed. The average forepaw distance was calculated by summing the distances for each step then dividing by the total number of steps. The number of unsuccessful forepaw steps divided by the total number of attempted forepaw steps was recorded as foot faults. An unsuccessful step was defined as an attempt to step or place the forepaw in which the paw clearly slipped from its destined position and had to be replaced on the grid by the animal.
Statistical analysis.
All cell culture experiments represent three to four independent experiments performed in triplicate. The in vivo studies represent 3–12 independent animals for each treatment. Data were calculated as mean ± standard error of the mean (SEM), and statistical analyses were performed on raw data. Differences between groups were determined by Student's t-test for analyses involving only two groups or by analysis of variance (ANOVA) followed by mean separation by the Student-Newman-Keuls (SNK) method for analyses involving more than two groups. Significance is reported at the level of p 
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MPP+ Toxicity is Prevented by Replacement of Complex I with Rotenone-Insensitive NDI1
In an earlier study, we demonstrated that stable expression of NDI1 in SK-N-MC neuroblastoma cells abolished rotenone toxicity, providing evidence of an obligatory role for complex I inhibition in rotenone toxicity (Sherer et al., 2003
). Here, we used the same cell line to determine whether complex I is required for MPP+ toxicity. NDI1 was properly incorporated into the mitochondria as evidenced by fluorescence micrographs demonstrating mitochondrial colocalization (far right panel) of NDI1 (left panel) and the F1-ATPase (middle panel) in SK-N-MC cells stably expressing NDI1 (Fig. 1A). These cells were then transfected with the human DAT to allow MPP+ to gain access to the cells (Richardson et al., 2005). Following exposure to 100µM MPP+ for 48 h, 50–60% cell death was observed and this value was set as the maximum % of cell death of 100%. However, cell death was totally abolished in cells overexpressing NDI1 (Fig. 1B), demonstrating the requirement of interaction of MPP+ with complex I to cause toxicity in this system. This effect appears to be dependent on the presence of NDI1 rather than altered uptake of the toxin, as there were no differences in 3H-MPP+ uptake between NDI1 cells and SK-N-MC cells transfected with the DAT (Fig. 1C).
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Viral-Mediated Expression of NDI1 In Vivo Attenuates MPTP Neurotoxicity
To confirm our in vitro findings, we employed a bilateral injection to deliver NDI1 in a viral vector to the brain, which we have previously demonstrated to confer expression in dopaminergic terminals in the striatum (Seo et al., 2004
). NDI1 expression did not alter basal dopamine levels when compared to animals which received vehicle at the time of surgery (data not shown) and these values were combined to form the control group. Administration of a moderate dose of MPTP (2 x 15 mg/kg) resulted in an 84% decrease of striatal dopamine 7 days after administration in animals receiving vehicle at surgery. This effect was greatly attenuated in mice expressing NDI1, with only a 48% decrease of striatal dopamine observed (Fig. 2A). There was a wide range of variance observed as some animals (n = 3) showed a small amount of protection (74% decrease of striatal dopamine), some (n = 6) that showed a moderate level of protection (60% decrease of striatal dopamine), and some (n = 3) that showed total protection as evidenced by no significant loss of dopamine. These results are consistent with variation in expression of NDI1 between animals and our previous observation that not all dopamine neurons express NDI1 after injection of rAAV-NDI1 (Seo et al., 2006). To confirm this variation, we attempted to perform immunohistochemical analysis of NDI1 expression in each individual animal. Unfortunately, technical limitations of the NDI1 antibody prevented successful quantification of NDI1 levels. However, overall significant neuroprotection was observed, suggesting that viral-mediated expression of NDI1 is a plausible means for providing neuroprotection against MPTP.
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In addition to measures of striatal dopamine depletion, we also assessed neurochemical markers in the striatum of these same animals to determine the degree of dopaminergic damage (Tillerson et al., 2002
). Similar to the effects observed on striatal dopamine depletion, NDI1 expression provided a significant level of neuroprotection against the loss of DAT (Fig. 2B), TH (Fig. 2C), and VMAT2 (Fig. 2D) protein levels as determined by immunoblotting. MPTP treatment caused a 70% reduction of DAT in animals receiving vehicle at surgery. This was significantly attenuated by NDI1, with only a 41% loss observed in animals receiving NDI1 at surgery. A similar protective effect was observed when we measured levels of TH and VMAT2. MPTP caused a 63 and 45% decrease in TH and VMAT2 levels in vehicle-injected mice. However, TH and VMAT2 levels were only decreased by 32 and 23% in NDI1-injected mice. As can be seen in the representative Western blots (Fig. 2E), the neuroprotection observed ranged from partial to complete. NDI1 expression did not alter DAT or VMAT2 levels as compared to animals that received vehicle at surgery (data not shown), suggesting that the neuroprotective effect of NDI1 against MPTP toxicity is likely not the result of altered uptake of or enhanced sequestration of MPP+. No significant effect of NDI1 expression was observed on TH protein levels. We observed a similar range of neuroprotection when TH immunoreactivity in the striatum was assessed by immunohistochemistry (Fig. 3A–F). Finally, we performed immunohistochemical staining of TH in the cell bodies in the substantia nigra pars compacta and found a similar range of neuroprotection (Fig. 3G–L). Although immunohistochemical staining as performed here is qualitative, these data support the ability of NDI1 to protect both the dopaminergic terminals and cell bodies from MPTP-induced neurodegeneration.
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Viral-Mediated Expression of NDI1 In Vivo Attenuates Glial and Microglial Activation
Astrogliosis and microgliosis have been used extensively as markers of MPTP-induced neurotoxicity (Francis et al., 1995
; O'Callaghan et al., 1990). Here, we determined GFAP levels as an indicator of astrogliosis (O'Callaghan et al., 1990) and Glut5 levels as an indicator of microgliosis (Payne et al., 1997) to determine whether NDI1 expression in vivo would attenuate the glial response in response to MPTP administration. Basal levels of GFAP and Glut5 as assessed by Western blot were not significantly different between animals administered vehicle and those administered NDI1 and were combined to form a pooled control group. MPTP treatment increased GFAP levels by 42% in animals receiving vehicle as the time of surgery, whereas those receiving NDI1 and subsequent MPTP treatment exhibited only a 17% increase in GFAP levels (Fig. 4A). A similar protective effect was observed for microglial proliferation as assessed by determining Glut5 levels. MPTP treatment increased Glut5 levels by 34% in animals receiving vehicle at surgery. This increase was attenuated in animals receiving NDI1, which only exhibited a 15% increase in Glut5 levels (Fig. 4B). As with the other measures of MPTP-induced neurotoxicity, there was a range of neuroprotection observed with these markers (Fig. 4C). However, NDI1 treatment provided a significant overall level of neuroprotection.
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Viral-Mediated Expression of NDI1 In Vivo Abolishes MPTP-Induced Behavioral Deficits
The loss of motor control and associated behavioral dysfunction is the most debilitating symptom of PD. In previous studies, we established the grid performance test as a sensitive indicator of dopamine-associated behavioral impairment in the MPTP mouse model of PD (Tillerson and Miller, 2003
; Tillerson et al., 2002). Here we sought to determine whether the amelioration of dopaminergic damage by NDI1 would result in prevention of MPTP-induced behavioral deficits. Administration of MPTP to animals given vehicle during surgery reduced forepaw step length on the grid to 50% of control values (Fig. 5) while animals expressing NDI1 did not exhibit a significant reduction in forepaw step length. In contrast to the variation in protective effect observed at the neurochemical level, NDI1-expressing animals were totally resistant to the behavioral deficit caused by MPTP neurotoxicity, suggesting that even a small amount of neuroprotection is sufficient to prevent behavioral impairment. NDI1 expression also decreased the percentage of foot faults in MPTP-treated animals but this effect did not quite reach statistical significance (p = 0.0579; data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There is accumulating evidence suggesting that oxidative damage to and dysfunction of complex I of the mitochondrial electron transport chain may be involved in PD pathogenesis (Dawson and Dawson, 2003
; Greenamyre et al., 2001). The observation of reduced complex I activity in PD patients provided some of the first clinical evidence of the potential involvement of complex I dysfunction in PD (Mizuno et al., 1989; Parker et al., 1989; Schapira et al., 1989). Most recently, Jin et al. (2006) reported three-fold decreases of two subunits of complex I in the substantia nigra of PD patients compared to age-matched controls using a proteomic approach. Furthermore, the development of the rotenone rat model of PD provided direct experimental evidence that complex I inhibition alone is sufficient to induce a parkinsonian syndrome (Betarbet et al., 2000). Although PD is not typically considered a mitochondrial disease, these studies cited above and recent work demonstrating that genes linked to PD affect mitochondrial function or localize to the mitochondria suggest that mitochondrial dysfunction, especially at complex I, may play a larger role in the development of PD than previously realized (Schapira, 2006).
In 1983, astute epidemiological and clinical observation by Langston et al. (1983) led to the identification of MPTP as a compound capable of causing an acute permanent parkinsonian syndrome that was virtually indistinguishable from PD in a group of drug users in California. Since this time, almost 4000 articles have been published on MPTP according to PubMed. However, the critical issue of how MPTP exerts its toxicity on dopamine neurons has remained a topic of debate. It has been argued that complex I inhibition does not completely explain the neurotoxicity of MPTP (Lotharius and O'Malley, 2000; Nakamura et al., 2000; Obata, 2002). Previously, we have demonstrated that MPP+ binds to the same site on complex I as rotenone (Richardson et al., 2005) and NDI1-expressing cells are resistant to rotenone toxicity because of the lack of this binding site (Sherer et al., 2003). Thus, NDI1-expressing cells provided a unique opportunity to define the role of interaction with complex I in the toxicity of MPP+. Here, we transfected the DAT into this cell line to directly determine the role of complex I in the toxicity of MPP+. MPP+ toxicity was totally abolished in the NDI1 cells, demonstrating that the lack of the binding site for rotenone on NDI1 was sufficient to prevent the toxicity of MPP+ and providing further support for our previous finding that the binding site for rotenone and MPP+ are the same (Richardson et al., 2005). Although these in vitro studies do not rule out additional downstream effects of MPP+ that may occur in vivo, such as microglial activation or excitotoxicity (Beal, 1998; Wu et al., 2002), these findings suggest that complex I is likely the primary molecular site of action and initiator of MPP+ neurotoxicity. An alternate hypothesis for MPTP toxicity has proposed that displacement of dopamine from synaptic vesicles (Lotharius and O'Malley, 2000; Obata, 2002) and resultant oxidative damage may be largely responsible for MPTP toxicity. However, a recent study employing genetic and pharmacological depletion of dopamine in mice revealed that the absence of dopamine did not exacerbate MPTP toxicity (Hasbani et al., 2005), suggesting that dopamine displacement does not play a primary role in MPTP toxicity and reinforcing findings that complex I is the primary molecular target of MPTP.
Having established that NDI1 overexpression prevented MPP+ toxicity in vitro, we next sought to determine whether bilateral injection of an adeno-associated virus carrying the NDI1 gene into the substantia nigra could prevent the neurotoxicity of MPTP. Recently, we utilized unilateral administration of NDI1 to test the ability of NDI1 to protect against high-dose MPTP toxicity and found some degree of neuroprotection for all but three of the 13 mice examined (Seo et al., 2006). Although the use of a unilateral injection paradigm in our initial study allowed the untreated side to be able to serve as an internal control, this may not be the most optimal paradigm for assessing the dopaminergic toxicity of MPTP. First, there appears to be a natural imbalance in the dopamine system between the left and right side of the caudate putamen. Indeed, unilateral toxin lesions in experimental animals can result in sprouting of dopaminergic projections on both sides, which may affect the outcomes measured (Blanchard et al., 1996). Here, we used bilateral injections of NDI1 and systemic MPTP to more closely mimic the clinical condition observed in PD. Bilateral injection of NDI1 into the substantia nigra did indeed provide significant protection against striatal dopamine depletion following MPTP administration (48% decrease in NDI1-expressing animals and 84% in non–NDI1-injected animals), similar to that observed with unilateral injection. Because we have previously observed that there appears to be variance in the level of transgene expression between individual animals (Seo et al., 2006), it is possible that there is variance in the degree of neuroprotection between individual animals. Indeed, we found that out of the 12 animals injected with NDI1 and MPTP, there was a wide range of protection observed (10–100%). These results were corroborated by Western blots of DAT, TH, and VMAT2 in these same animals, which we have previously shown to be excellent indicators of MPTP-induced dopaminergic damage (Tillerson et al., 2002). DAT levels were not significantly different between NDI1-expressing animals and those not expressing NDI1, suggesting that altered uptake of MPP+ is not likely to have influenced the results similar to what we observed in vitro. We also observed qualitatively similar results when we analyzed dopaminergic damage by immunohistochemical methods for TH in the dopaminergic cell bodies of the substantia nigra and terminals in the striatum. Finally, a similar degree of neuroprotection was observed when we assessed the degree of astrogliosis and microgliosis, which have been shown to be excellent indicators of MPTP-induced neurotoxicity and known to be increased in PD brain. It should be noted that we assessed astrogliosis and microgliosis at 7 days following injection, which is not the peak of activation of these markers (Francis et al., 1995). Therefore, we cannot rule out that similar degrees of glial activation may occur between vehicle and NDI1-injected animals treated with MPTP at the time of peak effect. Nonetheless, the totality of our assessments clearly demonstrates a significant protective effect of NDI1 against MPTP neurotoxicity. Although we did observe variance in the protective effect of NDI1, recent advances in viral and nonviral methods for introducing transgenes into the brain may provide an avenue for achieving increased and more consistent transgene expression allowing for increased neuroprotection in future experiments (de Lima et al., 2005).
In PD, there is evidence that neurodegeneration begins long before patients are diagnosed and that the behavioral symptoms that are characteristic of PD and the most devastating part of the disease process do not fully manifest until there is greater than 80% loss of striatal dopamine (Koller, 1992). We have previously developed sensitive behavioral tests in MPTP-treated mice that correlate highly with loss of striatal dopamine and are able to detect behavioral dysfunction when there is less than 80% dopamine depletion (Tillerson and Miller, 2003; Tillerson et al., 2002). Here, we observed total prevention of MPTP-induced behavioral deficits in NDI1-expressing mice. Significant protection was observed even in the animals that were only marginally protected from the neurochemical deficits caused by MPTP. These data suggest that even a small level of neuroprotection is sufficient to ameliorate behavioral deficits in an animal model of PD. Taken in concert, our data provide support for the inhibition of complex I as a major contributor to the dopaminergic degeneration and behavioral deficits observed in animals treated with MPTP.
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NOTES |
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1 Present address: The Michael J. Fox Foundation for Parkinson's Disease Research, New York, NY 10163.
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ACKNOWLEDGMENTS |
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This work was supported by NIH Grants U54 ES012068 as part of the Collaborative Centers for Parkinson's Disease Environmental Research (G.W.M. and J.T.G.), R01NS048441 and R01DK053244 (A.M.-Y. and T.Y.), F32ES013457 and T32NS07480 (J.R.R.), and T32ES012870 (W.M.C.). T.B.S. was supported by a Michael J. Fox Foundation Fellowship.
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Ali SF, David SN, Newport GD. (1991) Age-related susceptibility to MPTP-induced neurotoxicity in mice. Neurotoxicology 14:29–34.
Bai Y, Hajek P, Chomyn A, Chan E, Seo BB, Matsuno-Yagi A, Yagi T, Attardi G. (2001) Lack of complex I activity in human cells carrying a mutation in MtDNA-encoded ND4 subunit is corrected by the Saccharomyces cerevisiae NADH-quinone oxidoreductase (NDI1) gene. J. Biol. Chem. 276:38808–38813.
Beal MF. (1998) Excitotoxicity and nitric oxide in Parkinson's disease pathogenesis. Ann. Neurol. 44:S110–S114.[CrossRef][ISI][Medline]
Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT. (2000) Chronic systemic pesticide exposure reproduces features of Parkinson's disease. Nat. Neurosci. 3:1301–1306.[CrossRef][ISI][Medline]
Blanchard V, Anglade P, Dziewczapolski G, Savasta M, Agid Y, Raisman-Vozari R. (1996) Dopaminergic sprouting in the rat striatum after partial lesion of the substantia nigra. Brain Res. 709:319–325.[CrossRef][ISI][Medline]
Caudle WM, Richardson JR, Delea KC, Guillot TS, Wang M, Pennell KD, Miller GW. (2006) Polychlorinated biphenyl (PCB)-induced reduction of dopamine transporter expression as a precursor to Parkinson's disease associated-dopamine toxicity. Toxicol. Sci. 92:490–499.
Chen TS, Koutsilieri E, Rausch WD. (1995) MPP+ selectively affects calcium homeostasis in mesencephalic cell cultures from embryonal C57/Bl6 mice. J. Neural. Transm. 100:153–163.[CrossRef][ISI][Medline]
Dauer W and Przedborski S. (2003) Parkinson's disease: mechanisms and models. Neuron 39:889–909.[CrossRef][ISI][Medline]
Dawson TM and Dawson VL. (2003) Molecular pathways of neurodegeneration in Parkinson's disease. Science 302:819–822.
de Lima MC, da Cruz MT, Cardoso AL, Simoes S, de Almeida LP. (2005) Liposomal and viral vectors for gene therapy of the central nervous system. Curr. Drug Targets CNS Neurol. Disord. 4:453–465.[CrossRef][Medline]
Francis JW, Von Visger J, Markelonis GJ, Oh TH. (1995) Neuroglial responses to the dopaminergic neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mouse striatum. Neurotoxicol. Teratol. 17:7–12.[CrossRef][ISI][Medline]
Greenamyre JT, Sherer TB, Betarbet R, Panov AV. (2001) Complex I and Parkinson's disease. IUBMB Life 52:135–141.[ISI][Medline]
Hasbani DM, Perez FA, Palmiter RD, O'Malley KL. (2005) Dopamine depletion does not protect against acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in vivo. J. Neurosci. 25:9428–9433.
Jin J, Hulette C, Wang Y, Zhang T, Pan C, Wadhwa R, Zhang J. (2006) Proteomic identification of a stress protein, mortalin/mthsp70/GRP75: Relevance to Parkinson's disease. Mol. Cell. Proteomics 5:1193–1204.
Koller WC. (1992) When does Parkinson's disease begin? Neurology 42:27–31.[ISI][Medline]
Kooncumchoo P, Sharma S, Porter J, Govitrapong P, Ebadi M. (2006) Coenzyme Q(10) provides neuroprotection in iron-induced apoptosis in dopamine neurons. J. Mol. Neurosci. 28:125–141.[CrossRef][ISI][Medline]
Langston JW, Ballard P, Tetrud JW, Irwin I. (1983) Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis. Science 219:979–980.
Lotharius J and O'Malley KL. (2000) The parkinsonism-inducing drug 1-methyl-4-phenylpyridinium triggers intracellular dopamine oxidation. A novel mechanism of toxicity. J. Biol. Chem. 275:38581–38588.
Mizuno Y, Ohta S, Tanaka M, Takamiya S, Suzuki K, Sato T, Oya H, Ozawa T, Kagawa Y. (1989) Deficiencies in complex I subunits of the respiratory chain in Parkinson's disease. Biochem. Biophys. Res. Commun. 163:1450–1455.[CrossRef][ISI][Medline]
Nakamura K, Bindokas VP, Marks JD, Wright DA, Frim DM, Miller RJ, Kang UJ. (2000) The selective toxicity of 1-methyl-4-phenylpyridinium to dopaminergic neurons: The role of mitochondrial complex I and reactive oxygen species revisited. Mol. Pharmacol. 58:271–278.
Nicklas WJ, Vyas I, Heikkila RE. (1985) Inhibition of NADH-linked oxidation in brain mitochondria by 1-methyl-4-phenyl-pyridine, a metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine. Life Sci. 36:2503–2508.[CrossRef][ISI][Medline]
Obata T. (2002) Dopamine efflux by MPTP and hydroxyl radical generation. J. Neural. Transm. 109:1159–1180.[CrossRef][ISI][Medline]
Obata T. (2006) Nitric oxide and MPP(+)-induced hydroxyl radical generation. J. Neural. Transm. 113:1131–1144.[CrossRef][ISI][Medline]
O'Callaghan JP, Miller DB, Reinhard JF. (1990) Characterization of the origins of astrocyte response to injury using the dopaminergic neurotoxicant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Brain Res. 521:73–80.[CrossRef][ISI][Medline]
Olanow CW and Tatton WG. (1999) Etiology and pathogenesis of Parkinson's disease. Annu. Rev. Neurosci. 22:123–144.[CrossRef][ISI][Medline]
Parker WD, Boyson SJ, Parks JK. (1989) Abnormalities of the electron transport chain in idiopathic Parkinson's disease. Ann. Neurol. 26:719–723.[CrossRef][ISI][Medline]
Payne J, Maher F, Simpson I, Mattice L, Davies P. (1997) Glucose transporter Glut5 expression in microglial cells. Glia 21:327–331.[CrossRef][ISI][Medline]
Pifl C, Giros B, Caron MG. (1993) Dopamine transporter expression confers cytotoxicity to low doses of the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium. J. Neurosci. 13:4246–4253.[Abstract]
Ramachandiran S, Hansen JM, Jones DP, Richardson JR, Miller GW. (2006) Divergent mechanisms of paraquat, MPP+, and rotenone toxicity: Oxidation of thioredoxin and caspase-3 activation. Toxicol. Sci. 95:1163–171.
Ramsay RR, Krueger MJ, Youngster SK, Gluck MR, Casida JE, Singer TP. (1991) Interaction of 1-methyl-4-phenylpyridinium ion (MPP+) and its analogs with the rotenone/piericidin binding site of NADH dehydrogenase. J. Neurochem. 56:1184–1190.[CrossRef][ISI][Medline]
Richardson JR, Caudle WM, Wang MZ, Dean ED, Pennell KD, Miller GW. (2006) Developmental exposure to the pesticide dieldrin alters the dopamine system and increases neurotoxicity in an animal model of Parkinson's disease. FASEB J. 20:1695–1697.
Richardson JR and Miller GW. (2004) Acute exposure to aroclor 1016 or 1260 differentially affects dopamine transporter and vesicular monoamine transporter 2 levels. Toxicol. Lett. 148:29–40.[CrossRef][ISI][Medline]
Richardson JR, Quan Y, Sherer TB, Greenamyre JT, Miller GW. (2005) Paraquat neurotoxicity is distinct from that of MPTP and rotenone. Toxicol. Sci. 88:193–201.
Schapira AH. (2006) Mitochondrial disease. Lancet 368:70–82.[CrossRef][Medline]
Schapira AH, Cooper JM, Dexter D, Jenner P, Clark JB, Marsden CD. (1989) Mitochondrial complex I deficiency in Parkinson's disease. Lancet 1:1269.[ISI][Medline]
Seo BB, Kitajima-Ihara T, Chan EK, Scheffler IE, Matsuno-Yagi A, Yagi T. (1998) Molecular remedy of complex I defects: Rotenone-insensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae mitochondria restores the NADH oxidase activity of complex I-deficient mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 95:9167–9171.
Seo BB, Nakamaru-Ogiso E, Cruz P, Flotte TR, Yagi T, Matsuno-Yagi A. (2004) Functional expression of the single subunit NADH dehydrogenase in mitochondria in vivo: A potential therapy for complex I deficiencies. Hum. Gene. Ther. 15:887–895.[ISI][Medline]
Seo BB, Nakamaru-Ogiso E, Flotte TR, Matsuno-Yagi A, Yagi T. (2006) In vivo complementation of complex I by the yeast NDI1 enzyme: Possible application for treatment of Parkinson's disease. J. Biol. Chem. 281:14250–14255.
Seo BB, Wang J, Flotte TR, Yagi T, Matsuno-Yagi A. (2000) Use of the NADH-quinone oxidoreductase (NDI1) gene of Saccharomyces cerevisiae as a possible cure for complex I defects in human cells. J. Biol. Chem. 275:37774–37778.
Sherer TB, Betarbet R, Greenamyre JT. (2002) Environment, mitochondria, and Parkinson's disease. Neuroscientist 8:192–197.[Abstract]
Sherer TB, Betarbet R, Testa CM, Seo BB, Richardson JR, Kim JH, Miller GW, Yagi T, Matsuno-Yagi A, Greenamyre JT. (2003) Mechanism of toxicity in rotenone models of Parkinson's disease. J. Neurosci. 23:10756–10764.
Tillerson JL, Caudle WM, Reveron ME, Miller GW. (2002) Detection of behavioral impairments correlated to neurochemical deficits in mice treated with moderate doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Exp. Neurol. 178:80–90.[CrossRef][ISI][Medline]
Tillerson JL and Miller GW. (2003) Grid performance test to measure behavioral impairment in the MPTP-treated-mouse model of parkinsonism. J. Neurosci. Methods 123:189–200.[CrossRef][ISI][Medline]
Trevor AJ, Castagnoli N Jr,, Caldera P, Ramsay RR, Singer TP. (1987) Bioactivation of MPTP: Reactive metabolites and possible biochemical sequelae. Life Sci. 40:713–719.[CrossRef][ISI][Medline]
Vila M and Przedborski S. (2004) Genetic clues to the pathogenesis of Parkinson's disease. Nat. Med. 10:S58–S62.
Wu DC, Jackson-Lewis V, Vila M, Tieu K, Teismann P, Vadseth C, Choi DK, Ischiropoulos H, Przedborski S. (2002) Blockade of microglial activation is neuroprotective in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease. J. Neurosci. 22:1763–1771.
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