Parkinson's disease (PD) is a common neurodegenerative disorder that can be of either familial or non-familial (i.e. sporadic) etiology (Fahn and Przedborski 2000). Cardinal clinical features of PD include tremor, stiffness and slowness of movement, all of which are attributed to the dramatic loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) (Fahn and Przedborski 2000). Mutations in the presynaptic protein α-synuclein (α-syn) are associated with a familial form of PD (Polymeropoulos et al. 1997) that is clinically and pathologically indistinguishable from the most common sporadic form of this disabling neurodegenerative disorder. Mutant α-syn cytotoxicity is likely related to the fact that both of the identified point mutations may enhance the propensity of α-syn to interact with other intracellular proteins and increase its tendency to aggregate (Conway et al. 1998; El-Agnaf et al. 1998; Engelender et al. 1999; Giasson et al. 1999; Narhi et al. 1999). Although similar α-syn mutations are not found in sporadic PD (Golbe 1999), mounting evidence indicates that α-syn may also play a deleterious role in sporadic PD (Spillantini et al. 1997, 1998). Relevant to these observations, we have recently reported widespread nitration of proteins, which is a consequence of the reaction of nitrating agents such as peroxynitrite with proteins (Ischiropoulos and al Mehdi 1995), in Lewy bodies (LBs) in sporadic PD, in dementia with LBs (DLB) and in the LB variant of Alzheimer's disease (Duda et al. 2000a). Moreover, using specific monoclonal antibodies that recognize only nitrated α-syn, we have also demonstrated that α-syn is the major protein that is modified by nitration in the LBs of the above neurodegenerative disorders as well as in neuronal and glial cytoplasmic inclusions (GCIs) in multiple system atrophy (MSA) and in Hallervorden–Spatz disease (HSD) (Giasson et al. 2000a). These data provide, for the first time, compelling evidence for the presence of nitrative stress and formation of nitrating agents in human neurodegenerative disorders. They also substantiate the hypothesis that oxidative stress is a leading pathogenic mechanism in neurodegenerative disorders including PD (Przedborski and Jackson-Lewis 2000). In addition, exposure of wild-type human α-syn to nitrating agents in vitro causes nitration and cross-linking via the formation of dityrosine, recapitulating some aspects of the α-syn extracted from the brains of humans with α-syn aggregates (Duda et al. 2000b; Galvin et al. 2000; Giasson et al. 2000a). Extending these observations and consistent with the oxidative-nitrative stress hypothesis, we now present compelling evidence that α-syn is a specific target for tyrosine nitration in stably transfected cells challenged with peroxynitrite, and in mice intoxicated with the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tertahydropyridine (MPTP) (Przedborski et al. 2000a).
Structural and functional alterations of α-synuclein is a presumed culprit in the demise of dopaminergic neurons in Parkinson's disease (PD). α-Synuclein mutations are found in familial but not in sporadic PD, raising the hypothesis that effects similar to those of familial PD-linked α-synuclein mutations may be achieved by oxidative post-translational modifications. Here, we show that wild-type α-synuclein is a selective target for nitration following peroxynitrite exposure of stably transfected HEK293 cells. Nitration of α-synuclein also occurs in the mouse striatum and ventral midbrain following administration of the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Conversely, β-synuclein and synaptophysin were not nitrated in MPTP-intoxicated mice. Our data demonstrate that α-synuclein is a target for tyrosine nitration, which, by disrupting its biophysical properties, may be relevant to the putative role of α-synuclein in the neurodegeneration associated with MPTP toxicity and with PD.
substantia nigra pars compacta
Stable transfection of HEK 293 cells
Syn plasmids were constructed by inserting human α- or β-syn cDNAs (Jakes et al. 1994) into the mammalian expression vector pcDNA 3.1+ (Invitrogen, Carlsbad, CA, USA). HEK293 cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and cultured in 90% Dulbecco's modified Eagle's medium (high glucose), 10% fetal bovine serum, 2 mm l-glutamine and antibiotics. Cells were transfected with α-syn/pcDNA3.1+ or β-syn/pcDNA3.1+ using calcium phosphate precipitation buffered with N,N-bis[2-hydroxyethyl]-2-amino-ethanesulfonic acid (BES) (Chen and Okayama 1997). One day following transfection, the cells were re-plated on 10-cm dishes and selection with Geneticin (500 µg/mL) (Life Technologies, Rockville, MD, USA) was initiated 24 h later. Individual stable clones were isolated with glass cylinders and detached from the dish with trypsin. Stable clones were re-plated and maintained in culture medium with Geneticin. Clones expressing high levels of syn were screened by western blot analysis.
Exposure of cells to peroxynitrite
Cells re-plated on six-well dishes in fresh culture medium were exposed to peroxynitrite by the addition of small drops above the cell surface and followed by rapid mixing to give a final concentration of 1 mm. The concentration of peroxynitrite was measured by the increase in absorbance at 302 nm in 1.2 m NaOH as described previously (Ara et al. 1998). The pH of the buffer was measured at the end of the exposure and was the same as before each addition of peroxynitrite. For all experiments, at the end of the incubation period, cells were washed with Earle's balanced salt solution, scraped-off of the plates and centrifuged at 8000 g for 5 min. The pellet was solubilized with lysis buffer [20 mm Tris-HCl (pH 7.4), 150 mm NaCl, 4 mm EGTA, 10% glycerol, 1% Triton-X 100]. As a control, peroxynitrite was allowed to decompose in cell media prior to exposure to the cells. This experiment controlled for hydrogen peroxide and nitrite, which are found in peroxynitrite solutions.
Mice and MPTP administration
Eight-week-old male C57/bl mice (22–25 g, Charles River Breeding Laboratories, Wilmington, MA, USA) were used. Animals were housed with three per cage in a temperature-controlled room under a 12-h light/12-h dark cycle with free access to food and water. Mice used in this study were treated according to the NIH guidelines for Care and Use of Laboratory Animals and with the approval of Columbia University's Institutional Animal Care and Use Committee. On the day of the experiment, mice received four i.p. injections of MPTP-HCl (20 mg/kg) in saline at 2 h intervals and were killed at 4 and 24 h post-injection; control mice received saline injections only. MPTP use and safety precautions were as described previously (Przedborski et al. 2000b). Frozen mouse striatal and ventral midbrain samples were homogenized (Polytron) in 5.0 mL of extraction buffer (phosphate-buffered saline containing 1 mm phenylmethylsulfonyl fluoride and aprotinin at 10 mg/mL) at 4°C.
Immunoprecipitation and western blotting
Solubilized extracts from cells and mice were briefly sonicated and centrifuged (325 g for 5 min) to remove cellular debris. Protein concentration was determined using a BCA kit (Pierce, Rockford, IL, USA). Solubilized proteins (2 mg) were precleared (45 min, 4°C) with 15 µL of Gamma bind plus Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and the supernatant was incubated (16 h, 4°C) with a rabbit anti-3-nitrotyrosine (3-NT) polyclonal antibody previously characterized (Beckman et al. 1994; Ye et al. 1996), or a mouse anti-α-syn monoclonal antibody (Syn-1) (Transduction Laboratories, Lexington, KY, USA). Immune complexes were absorbed (1.5 h, 4°C) to 50 µL of Gamma Bind plus Sepharose, extensively washed with extraction buffer by sequential sedimentation and resuspension, eluted in sample loading buffer by heating to 95°C for 5 min, and resolved by SDS–PAGE on 12–15% polyacrylamide gels. Proteins were electrophoretically transferred to nitrocellulose or PVDF membranes, which were blocked with 5% non-fat dry milk in 1 × TBS, 0.1% Tween 20 for 1 h. Incubation with one of the primary antibodies was performed overnight at 4°C using either Syn-1, anti-β-syn (a gift from Dr S. Nakajo, Tokyo, Japan), Syn 207 (Giasson et al. 2000b), anti-synaptophysin (a gift from Dr Honer, Albert Einstein College of Medicine, NY, USA) or anti-3-NT antibody. Incubation with a secondary anti-mouse- or anti-rabbit-conjugated horseradish peroxidase antibody was performed at 25°C for 1 h. After washing in 1 × TBS, 0.1% Tween-20, blots were developed with Super Signal Ultra chemiluminiscence (Pierce) and exposed to Kodak BetaMax film.
Results and discussion
In the present study, we demonstrate that α-syn is a specific target for tyrosine nitration in a cell model as well as in the mouse brain after MPTP intoxication. First, non-transfected HEK293 cells and HEK293 cells overexpressing human α-syn or β-syn were exposed to peroxynitrite, an oxidizing and nitrating agent (Ischiropoulos and al Mehdi 1995). This treatment resulted in the nitration of a number of proteins as demonstrated by the western blot analysis using an anti-NT antibody (Fig. 1a). However, only HEK293 cells transfected with α-syn showed a nitrated protein band with the molecular mass corresponding to α-syn. To demonstrate that α-syn was indeed nitrated, the total protein extract was subjected to immunoprecipitation using an anti-α-syn antibody and then the recovered immunoprecipitated protein was probed with the anti-3-NT antibody. This experiment confirmed that a significant fraction of the immunoprecipitated α-syn was definitely nitrated in the cells exposed to peroxynitrite (Fig. 2, lane 3), but not in untreated cells or in cells exposed to decomposed reagent (Fig. 2, lanes 1 and 2).
Given these results, we then proceeded to assess whether α-syn was nitrated in the MPTP mouse model of PD. The use of this particular experimental model has been motivated by the fact that, thus far, significant insights into the pathogenesis of PD have been achieved using this neurotoxin, which replicates in humans and in non-human primates a severe and irreversible PD-like syndrome, with concomitant degeneration of dopaminergic neurons (Przedborski et al. 2000a). Moreover, several studies have indicated that reactive nitrogen species and tyrosine nitration not only occur in this model but also participate in the MPTP neurotoxic process (Schulz et al. 1995; Przedborski et al. 1996; Ara et al. 1998; Mandir et al. 1999; Pennathur et al. 1999).
Immunoprecipitation of α-syn was performed as above from striatum and ventral midbrain, the two main targets of MPTP neurotoxicity (Przedborski et al. 2000a). Immunoprecipitated α-syn from striatum and ventral midbrain was selectively nitrated 4 h after the MPTP administration (Fig. 3). Conversely, immunoprecipitation of α-syn from striatum and ventral midbrain of saline-injected mice, also at 4 h post-injection, did not reveal any detectable nitration of the protein (Fig. 3). In contrast to the robust tyrosine nitration of α-syn (Figs 3a and b), no tyrosine nitration was detected in two other presynaptic proteins, β-syn and synaptophysin, following a similar MPTP administration (Figs 3c and d). This observation is consistent with our previous finding that only selected proteins are tyrosine-nitrated after MPTP exposure (Ara et al. 1998; Ischiropoulos 1998) and with the observation that α-syn, but not β-syn, is nitrated after exposure of cells to the same peroxynitrite challenge. Moreover, this observation is consistent with the demonstrations that nitrated α-syn is present in the hallmark lesions in a number of human neurodegenerative synucleinopathies (Duda et al. 2000a; Giasson et al. 2000a).
The higher efficiency of α-syn nitration is likely to be caused by the unstructured conformation of the protein in aqueous solution, which exposes all four tyrosine residues to the solvent phase and increases the probability of the reaction with nitrating agents. Moreover, glutamate residues, a structural conformation associated with enhanced susceptibility of tyrosine to nitration, are near all three tyrosine residues (125, 133 and 136) in the carboxy terminal domain of α-syn. Indeed, purified human α-syn exposed to the nitrating agent in vitro and analyzed by electrospray mass spectrometry, revealed that nitration of α-syn occurs at all four tyrosine residues (Souza et al. 2000). In addition to nitration, exposure of α-syn to nitrating agents also results in the stable cross-linking of the protein via the formation of dityrosine (Souza et al. 2000). In contrast, β-syn is nitrated to a lesser extent than α-syn in vitro and does not form stable O-O′dityrosine crosslinks after exposure to nitrating agents, despite the presence of all four conserved tyrosine residues in both proteins (Souza et al. 2000). More significantly, we show here that in two in vivo models, α-syn is selectively nitrated, whereas nitration of β-syn is below detectable limits. The preferential nitration and oxidation of tyrosine residues in α-syn could be caused by the accessibility of tyrosine residues to nitrating agents and by the presence of the protein in close proximity to the site(s) of generation of the nitrating agent. Our results raise the possibility that both syn proteins may have different confirmations, or that β-syn may be protected from oxidation, perhaps by different interacting partners in vivo. We have previously argued that proximity to sites of superoxide generation may be important in determining proteins modified by nitration, as overexpression of superoxide dismutase and superoxide mimetics have been shown to prevent the nitration of proteins in vivo and in cell models (Ara et al. 1998; Cuzzocrea et al. 2000; Pong et al. 2000).
The significance of the tyrosine nitration of α-syn remains unclear. Tyrosine nitration induces secondary and tertiary structural alterations, which may critically modify protein functions (Ischiropoulos 1998). The change in the ionization state of the modified protein induced by a local shift in the pKa from 10.01 of tyrosine to 7.5 of 3-NT, and the consequent changes in hydrophobicity and conformation, may facilitate interactions with other proteins, thereby promoting protein aggregation. Preliminary data indeed indicate an increased adherence of mouse brain extracts to nitrated α-syn compared with the unmodified wild-type protein (Chen et al., unpublished observation).
Collectively, the data indicate that α-syn is a preferential target for oxidative stress-mediated post-translational modifications. These alterations may trigger abnormal protein compartmentalization and aggregation, two phenomena that are potential culprits for the neurodegeneration process in PD.
This study was supported by the NIH-NINDS and NIH-NIA grants (SP, VMY-L and HI), the Department of Defense DAMD 17–99–1-9474, the Parkinson's Disease Foundation, the Lowenstein Foundation, the Smart Foundation, the Muscular Dystrophy Association, the ALS Association and the Project-ALS (SP). MV and BIG are recipients of a Fellowship from the Human Frontier Science Program Organization.