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α-Synuclein expression is increased in dopaminergic neurons challenged by toxic insults. Here, we assessed whether this upregulation is accompanied by pathological accumulation of α-synuclein and protein modifications (i.e. nitration, phosphorylation and aggregation) that are typically observed in Parkinson disease and in other synucleinopathies. A single injection of the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to squirrel monkeys caused a buildup of α-synuclein but not of β-synuclein or synaptophysin within nigral dopaminergic cell bodies. Immunohistochemistry and immunoelectron microscopy (IEM) also revealed large numbers of dystrophic axons labeled with α-synuclein. Antibodies that recognize nitrated and phosphorylated (at serine 129) α-synuclein stained neuronal cell bodies and dystrophic axons in the midbrain of MPTP-treated animals. Following toxicant exposure, α-synuclein deposition occurred at the level of neuronal axons in which amorphous protein aggregates were demonstrated by IEM. In a subset of these axons, immunoreactivity for α-synuclein was evident after tissue digestion with proteinase K, further indicating the accumulation of insoluble protein. These data indicate that toxic injury can induce the α-synuclein modifications that have been implicated in the pathogenesis of human synucleinopathies. The findings are also consistent with a pattern of evolution of α-synuclein pathology that may begin with the accumulation and aggregation of the protein within damaged axons.
The discovery that point mutations in the gene encoding α-synuclein (SNCA) are associated with familial parkinsonism provided the first evidence of a pathogenetic role of this protein in human neurodegenerative diseases (1-3). Subsequent studies demonstrated that the involvement of α-synuclein in pathologic processes is not limited to rare familial cases of parkinsonism and is not necessarily dependent on mutated forms of the protein. α-Synuclein is a major component of the intraneuronal inclusions called Lewy bodies and Lewy neurites that are typically observed in all patients with idiopathic Parkinson disease (PD). α-Synuclein inclusions are also found in other neurodegenerative diseases including dementia with Lewy bodies and multiple system atrophy (4, 5). The vast majority of patients affected by these diseases do not carry any α-synuclein mutations, indicating that toxic properties inherent to normal (i.e. non-mutated) α-synuclein are likely to underlie its role in disease pathogenesis (6-8). Furthermore, deleterious effects of normal α-synuclein are clearly supported by relationships between SNCA genomic multiplication, enhanced expression of the wild-type protein, and the development of familial parkinsonism (9, 10). The latter relationship is of particular relevance since it suggests that any condition capable of augmenting α-synuclein levels may contribute to the pathogenesis of PD and other synucleinopathies.
Vila and colleagues (11) were the first to report an intriguing relationship between toxicant-induced neuronal damage and α-synuclein upregulation in an animal model of PD. After five consecutive daily injections of 1-methyl-4-pheny-1,2,3,6-tetrahydropyridine (MPTP), nigrostriatal degeneration was accompanied by a significant increase in α-synuclein mRNA and protein in mouse midbrain extracts. Protein level reached its maximum a few days after exposure and returned to normal values by day 7 post-MPTP. Subsequent studies have demonstrated that α-synuclein upregulation is not a unique feature of the MPTP model of dopaminergic cell death. Indeed, it has also been described after treatment of rodents with other neurotoxicants that preferentially damage the nigrostriatal system such as paraquat and methamphetamine (12-14). Taken together, these findings suggest that enhanced α-synuclein expression is a common neuronal response to injury. They are also consistent with the possibility that, given the harmful consequences of excessive α-synuclein, this response could play an important role in pathogenetic processes underlying human neurodegenerative diseases.
A potential link between toxicant exposure and α-synuclein alterations is further supported by studies in non-human primates. MPTP administration was found to cause a sustained (i.e. at least one month) upregulation of α-synuclein in squirrel monkeys (15) and accumulation of the protein within dopaminergic cell bodies in both squirrel monkeys and baboons (15, 16). Kowall and colleagues proposed that this latter effect may model the early stages of Lewy body formation (16). To date, however, no direct evidence supports the deposition of insoluble, aggregated α-synuclein in MPTP-exposed monkeys. Furthermore, other protein modifications (i.e. nitration and phosphorylation) that are typical of Lewy bodies in human synucleinopathies have not been described in the primate MPTP model.
In the present study, changes in α-synuclein expression caused by MPTP were correlated with nitration, phosphorylation and aggregation of the protein in the monkey midbrain. The data reveal significant protein abnormalities at the level of both neuronal cell bodies and neuritic processes; the latter showed not only accumulation of nitrated and phosphorylated α-synuclein but also formation of α-synuclein aggregates.
A total of 11 feral middle-aged squirrel monkeys (Saimiri sciureus) of both sexes were obtained from Osage Research Primates (Osage Beach, MO). The animals were individually housed in a room with a 13/11-hour light/dark cycle, with free access to water and a daily diet of monkey chow and fresh fruit. Monkeys received a single subcutaneous injection of either saline (n = 4) or MPTP (Sigma, St. Louis, MO) at a dose of 1.75 mg/kg (n = 7). One month later, the animals were killed according to the recommendations of the Panel of Euthanasia of the American Veterinary Medical Association. All experimental protocols were in accordance with the standards established by the National Institutes of Health and the Office of the Prevention of Research Risks and were approved by the Institutional Animal Care and Use Committee.
Brains were rapidly removed and dissected sagittally along the midline. A tissue block containing the midbrain from the right hemisphere was fixed in 4% paraformaldehyde for 3 days and 10% formalin for 10 days. It was then cryoprotected and frozen on dry ice. Serial coronal sections 40-μm thick were collected throughout the substantia nigra and stored in cryopreservative for immunohistochemistry as previously described (17). A midbrain block was also dissected from the left hemisphere and sectioned into 1-mm coronal slices. These slices were fixed in 10% formalin for two days, and small cores of tissue were taken from the dorsomedial ventral midbrain with a tissue punch and processed for electron microscopy as previously described (18).
One set of sections was immunostained for bright-field microscopy. After washing in 0.01 M phosphate buffered saline (pH 7.4, PBS), endogenous peroxidase was quenched by incubation in hydrogen peroxide solution. Sections were blocked in 10% normal serum and then incubated overnight at 4°C in the following primary antibodies diluted in PBS: mouse anti-α-synuclein (1:600, Lab Vision, Fremont, CA), mouse anti-nitrated α-synuclein (1:4,000, Millipore, Billerica, MA), mouse anti-phospho-Ser 129 α-synuclein (1:3,000, gift from Elan Pharmaceuticals, South San Francisco, CA), sheep anti-β-synuclein (1:600, Chemicon, Temecula, CA) or mouse anti-synaptophysin (1:1,000, Chemicon). Immunolabeling was visualized using the ABC peroxidase method with 3,3'-diaminobenzidine (DAB) as the chromogen (Vector Laboratories, Burlingame, CA). Sections were lightly counterstained with Cresyl violet (FD Neurotechnologies, Ellicott City, MD), dehydrated and mounted in Depex mounting medium (E.M. Sciences, Hatfield, PA). For immunolabeling with anti-phospho-Ser 129 α-synuclein, sections were incubated in 30% formic acid for 30 seconds, rinsed in PBS and then processed as described above (19).
Proteinase K pre-treatment was performed on tissue sections that had been mounted onto glass Superfrost slides (Fisher Scientific, Pittsburgh, PA) and allowed to dry overnight. Sections were incubated at 55°C in 50 μg/ml proteinase K (Invitrogen, Carlsbad, CA) for 60 minutes, washed in PBS, and then immunostained for α-synuclein using the protocol described above. Concentration of proteinase K, length of incubation and temperature of incubation were chosen based on preliminary experiments showing that these conditions achieved maximum digestion of α-synuclein in control tissues (no detectable α-synuclein immunoreactivity) without causing significant tissue damage (i.e. extending the incubation time produced vacuolization and tearing of the sections). The protocol of proteinase K treatment is similar to that described in earlier reports in which the presence of soluble versus insoluble α-synuclein was assessed in free-floating tissue sections from the monkey midbrain (20, 21).
A second set of sections was dual-labeled with a combination of antibodies for confocal microscopy. Tissues were blocked in 10% normal donkey serum and placed in the following primary antibodies overnight at 4°C: sheep anti-α-synuclein (1:600, Chemicon), rabbit anti-GFAP (glial fibrillary acidic protein, 1:600, Dako, Carpinteria, CA), rabbit anti-Iba1 (ionizing calcium-binding adaptor molecule 1, 1:1,000; Biocare Medical, Concord, CA), mouse anti-nitrated α-synuclein (1:4,000, Millipore) or mouse anti-phospho-Ser 129 α-synuclein (1:3,000, gift from Elan Pharmaceuticals). Sections were then incubated with the appropriate fluorescent secondary antibody conjugated to either FITC or Cy-3 (1:500; Jackson ImmunoResearch Laboratories, West Grove, PA). Tissues immunostained for phospho-Ser 129 α-synuclein were pre-treated in formic acid as described above. Sections were mounted in Vectorshield Hardset mounting medium (Vector Labs) and observed using the Zeiss Pascal LSM confocal microscopy system. For both bright field and confocal microscopy, control sections incubated in the appropriate IgG were devoid of staining.
Tissues were embedded in LR White resin, and thin sections prepared and collected onto formvar-coated copper grids. The sections were blocked for 15 minutes in 5% normal goat serum and incubated in mouse anti-α-synuclein (1:10; Lab Vision). Grids were then immersed in a goat anti-mouse secondary antibody conjugated to 10-nm gold particles (1:50; Ted Pella, Redding, CA). Grids were post-fixed in 8% glutaraldehyde (Ted Pella) for 10 minutes and dried for 30 minutes. Sections were counterstained with uranyl acetate and lead citrate prior to viewing on a JEOL 1230 transmission electron microscope.
Tissues from three control monkeys and three animals injected with MPTP were used for this analysis. For each animal, a midbrain section at the level of the third nerve was immunostained for α-synuclein (bright-field microscopy). The ventral midbrain was delineated at low magnification (×1.25) as the area ventral to the central grey substance, lateral to the midline, medial to the outer edge of the substantia nigra pars lateralis and dorsal to the cerebral peduncle. The entire area was systematically examined at high magnification (×60) and α-synuclein-positive dystrophic axons were counted. Counts were carried out using the meander tool of the StereoInvestigator software (MBF Bioscience, Williston, VT).
A single subcutaneous injection of 1.75 mg/kg MPTP causes a 40% loss of nigral dopaminergic neurons in squirrel monkeys (15). It also induces upregulation of α-synuclein that is sustained throughout the 4-week period during which nigrostriatal degeneration progresses in this model (15). The first set of experiments was designed to assess whether increased protein levels represent a response to injury unique to α-synuclein or common to other neuronal proteins. Midbrain sections from monkeys killed at one month after saline or MPTP treatment were immunostained with antibodies against α-synuclein, β-synuclein or synaptophysin. The rationale for comparing the effects of MPTP on these three proteins is twofold. First, α- and β-synuclein are members of the same family of proteins that share sequence and structural homologies (22). Second, α-synuclein, β-synuclein and synaptophysin are all synaptic proteins that could presumably undergo similar changes after toxicant-induced neuronal injury (23). In tissues from control monkeys immunostained for α-synuclein, β-synuclein or synaptophysin, immunoreactivity labeled the neuropil in a punctuate pattern, with little or no staining of neuronal cell bodies (Fig. 1A, C, E). In MPTP-treated animals, neuromelanin-containing cells (i.e. dopaminergic neurons) displayed robust α-synuclein immunoreactivity (Fig. 1B). MPTP treatment, however, did not enhance β-synuclein or synaptophysin immunostaining nor did it change their distribution between synaptic profiles and neuronal cell bodies (Fig. 1D, F).
In addition to increased α-synuclein immunoreactivity within neuronal cell bodies, MPTP-treated but not control monkeys (Fig. 2A, B) had enlarged dystrophic axons that stained for α-synuclein. There was widespread occurrence of this abnormality as shown in a low magnification image of the ventral midbrain from an MPTP-treated monkey (Fig. 2B). Swollen axons varied in size (i.e. up to 20 μm in diameter) and morphology; they had features similar to α-synuclein-positive neurites in PD midbrain (Fig. 2C, D). They were not evenly distributed across the region but were more concentrated along the dorso-medial tier of the substantia nigra and the ventral portion of the ventral tegmental area. This pattern most likely reflects the topographical distribution of the ascending projections of nigral dopaminergic neurons (24). The specificity of axonal α-synuclein pathology was indicated by lack of axonal α-synuclein or synaptophysin immunoreactivity (data not shown).
Neuritic accumulation of α-synuclein was confirmed by IEM. α-Synuclein labeling was increased within myelinated as well as unmyelinated neurites (axons and dendrites) in MPTP-(Fig. 3B) compared to saline-treated (Fig. 3A) monkeys. In some instances, MPTP-injured neurites were characterized by the clustering of gold particles that formed bundles of amorphous deposits (Fig. 3B, inset). Figure 3C shows the normal ultrastructural features of midbrain tissue from a saline-injected monkey. Following MPTP administration, enlarged myelinated axons labeled with α-synuclein were frequently present in IEM sections (Fig. 3D, E); these most likely correspond to the dystrophic axons observed by light microscopy.
Accumulation of α-synuclein within non-neuronal cells, including astrocytes and oligodendroglia, has been described in PD, multiple system atrophy and other synucleinopathies (5, 25, 26). Furthermore, experimental data indicate that α-synuclein can be taken up by microglia and that this phagocytosis may lead to microglial activation (27, 28). Since MPTP neurotoxicity is accompanied by marked astrocytosis and microglial activation (29, 30), we assessed whether α-synuclein immunoreactivity colocalized with markers of astrocytes (GFAP) and/or microglia (Iba1) in midbrain sections from control and MPTP-treated monkeys. Confocal images showed no overlapping signals in saline-injected animals (data not shown). Similarly, tissues from MPTP-treated animals showed a distinct cellular distribution of α-synuclein compared to GFAP and Iba1 immunoreactivities and there was no evidence of α-synuclein accumulation within astrocytes or microglia (Fig. 4).
Antibodies that specifically recognize tyrosine-nitrated epitopes of α-synuclein have been shown to label Lewy bodies and Lewy neurites in PD and other synucleinopathies (31). To determine whether accumulation of α-synuclein within neurons of MPTP-treated monkeys was accompanied by protein nitration, midbrain sections were immunostained with an anti-nitrated α-synuclein antibody (31). No specific labeling was detected in sections from saline-treated control animals (Fig. 5A). In contrast, neuromelanin-containing cell bodies and dystrophic neurites immunoreactive for nitrated α-synuclein were scattered throughout the midbrain of MPTP-injected monkeys (Fig. 5B, C). To assess the extent of α-synuclein nitration further, tissues were double-labeled with the anti-nitrated α-synuclein antibody together with an antibody against unmodified α-synuclein. The results indicated that only a small percentage of neuronal cell bodies (not shown) and axons (Fig. 5D-F) that were immunoreactive for unmodified α-synuclein were also labeled for the nitrated form of the protein.
Another post-translational modification of α-synuclein associated with human synucleinopathies is phosphorylation at Ser129 (32). Using an antibody that specifically recognizes phospho-Ser 129 α-synuclein (19), immunoreactive dopaminergic cell bodies were frequently observed in midbrain sections of MPTP-treated but not control animals (Fig. 6A-C). Phospho-Ser 129 α-synuclein was also present within some but not all dystrophic axons. These neurites could be double labeled using an antibody against unmodified α-synuclein and the anti-phospho-Ser 129 α-synuclein antibody (Fig. 6D-F).
IEM observations provided initial evidence of α-synuclein deposition within neurites of MPTP-challenged monkeys (see above, Fig. 3B). Further evaluation of protein aggregation was carried out by treating midbrain tissue sections with proteinase K. This treatment completely digests soluble α-synuclein, whereas insoluble (aggregated) forms of the protein are resistant to enzymatic cleavage (21, 33). Proteinase K abolished α-synuclein immunostaining in sections from saline-injected control animals (Fig. 7A, B). It also digested α-synuclein within neuromelanin-containing neurons of MPTP-exposed monkeys, indicating that soluble rather than aggregated α-synuclein is accumulated within injured nigral neuron cell bodies (Fig. 7C, D). In contrast, treatment with proteinase K reduced but did not completely eliminate α-synuclein immunoreactivity associated with dystrophic axons (Fig. 7E, F), suggesting the presence of both soluble and insoluble protein.
The number of enlarged α-synuclein-labeled axons was counted in two sets of sections of the ventral midbrain at the level of the third cranial nerve from both saline- and MPTP-injected monkeys. One set was untreated and the other set was incubated with proteinase K prior to α-synuclein immunostaining. No pathologic profiles were observed in either untreated or proteinase K-digested sections from control animals. Untreated sections from MPTP-injected monkeys contained 158 ± 76 dystrophic axons; this number was reduced to 23 ± 4 (mean ± SD) by proteinase K digestion. Thus, following toxicant exposure, aggregated protein was present in approximately 15% of α-synuclein-immunoreactive dystrophic axons.
Administration of MPTP to non-human primates has been widely used for investigation into mechanisms of nigrostriatal degeneration but the relationship between toxicant exposure and pathologic alterations of α-synuclein remains relatively unexplored in this model. Previous studies have shown MPTP-induced changes in α-synuclein expression and intraneuronal distribution. Purisai et al reported increased levels of α-synuclein mRNA and protein in squirrel monkeys at one week and one month after a single injection of MPTP (15). At the later time point, α-synuclein immunoreactivity, which normally labels synaptic profiles and neuronal fibers, robustly stained dopaminergic cell bodies. In a separate study using baboons that were repeatedly injected with MPTP, Kowall and colleagues also found a pronounced redistribution of α-synuclein that appeared to cluster within neuronal somata in the form of α-synucleinimmunoreactive granules ten days after the initial MPTP administration (16). Taken together, evidence from these reports indicates that increased levels of α-synuclein and protein accumulation in nigral neuron cell bodies represent important outcomes of MPTP-induced neuronal injury that occur in different primate species and under different MPTP regimens.
Our current results reveal a number of other important features of the primate MPTP model concerning α-synuclein modifications. We found that protein elevation only occurs within neuronal cells since no α-synuclein immunoreactivity labeled astrocytes or microglia. Our data also indicate that increased expression is relatively selective for α-synuclein, since immunoreactivity for other synaptic proteins (β-synuclein and synaptophysin) did not differ between control and MPTP-treated monkeys. Quite importantly, by the use of bright-field, confocal and IEM analyses, we document for the first time that the extensive neuritic pathology caused by MPTP involves the accumulation of α-synuclein within enlarged dystrophic axons. Several mechanisms are likely to contribute to this abnormal α-synuclein buildup. Since administration of MPTP to squirrel monkeys elevates levels of α-synuclein mRNA and protein (15), increased α-synuclein synthesis in neuronal perikarya would be followed by axonal transport of the protein to its presynaptic site (34, 35). This transport is likely to be impaired by MPTP-induced injury of neuronal terminals (36-38) and could be further disrupted by the formation of axonal α-synuclein aggregates (see below). Thus, the combination of protein upregulation, toxic lesion of the terminals and obstructive deposition could contribute to the axonal accumulation of α-synuclein observed in this model.
Antibodies that recognize tyrosine-nitrated epitopes of α-synuclein have been reported to label Lewy bodies and Lewy neurites in PD and other neurodegenerative synucleinopathies (31). Since protein nitration is likely a consequence of intraneuronal production of superoxide, nitric oxide and peroxynitrite, this modification of α-synuclein has been suggested to be a marker of oxidative processes that underlie disease pathogenesis (31, 39). Oxidative and nitrative damage also accompanies dopaminergic cell degeneration after MPTP exposure (40-42) and this likely explains the formation of nitrated α-synuclein within midbrain neurons of our MPTP-treated monkeys. Whether α-synuclein nitration is a mere sign of oxidative reactions or represents an important step toward the development of α-synuclein pathology (e.g. protein deposition) remains uncertain (39, 43). The pathological implications of another post-translational modification of α-synuclein (i.e. phosphorylation at Ser129) are relatively clearer. This single phosphorylation has been shown to be the predominant modification of α-synuclein in Lewy bodies in human synucleinopathies (19, 32). α-Synuclein phosphorylation is catalyzed by specific kinases, including casein kinase 1 and 2 and G-protein coupled kinases, and can be triggered, at least in vitro, by oxidative challenges (44-47). Perhaps most importantly, phosphorylation at Ser129 modifies the biological/toxic properties of α-synuclein, enhancing, for example, its tendency to aggregate, thereby reducing its binding to membrane phospholipids and altering its interactions with other proteins (32, 45, 46, 48). Our current study reveals that phospho-Ser 129 α-synuclein is formed as a consequence of neuronal injury in the primate MPTP model. This finding is likely to have significant implications since the presence of phosphorylated α-synuclein (i) could be a key event in the development of further α-synuclein pathology (19), and (ii) may indicate a gain of toxic function of the protein that could contribute to neurodegenerative processes.
Although no Lewy body-like inclusions were observed within dopaminergic cells of monkeys treated with MPTP, the present study showed evidence of α-synuclein aggregation. Treatment with proteinase K revealed the presence of insoluble α-synuclein in midbrain tissue from MPTP-exposed animals and IEM confirmed the intraneuronal aggregation of α-synuclein into amorphous bundles. Interestingly, both proteinase K-resistant aggregates and α-synuclein-positive deposits were observed at the level of neuronal axons rather than cell bodies. This pattern of protein deposition suggests that MPTP-induced α-synuclein pathology primarily affects damaged axons. Our present observations are also consistent with the possibility that obstructive α-synuclein axonal deposition may contribute to a retrograde buildup of the protein that, unlike its normal distribution, becomes highly concentrated into neuronal cell bodies of MPTP-treated monkeys (15, 16).
Forno and colleagues (49) have described the formation of eosinophilic Lewy body-like inclusions in squirrel monkeys injected with MPTP. This observation appears to be at odds with results of the current study in which we failed to identify organized Lewy body-like structures in MPTP-treated non-human primates. Differences in experimental protocols, however, likely explain these inconsistencies. In the earlier report, monkeys received repeated MPTP injections over a protracted period of time whereas, in the present investigation, they were subjected to a single toxic exposure. Furthermore, Forno and colleagues (49) observed intraneuronal inclusions only in old animals, whereas the present experiments were done with middle-aged squirrel monkeys. Taken together, previous and current findings indicate that, besides the toxic insult, other important factors, such as time post-injury, repeated challenges and aging, play a critical role in the development of α-synuclein inclusions after the initial protein aggregation. A contribution of aging is particularly intriguing in view of the fact that levels of α-synuclein have been reported to increase with normal aging in the primate substantia nigra (21, 50).
The experimental protocol of MPTP administration used in the present study causes significant neurodegeneration in the monkey substantia nigra (15). Changes in α-synuclein expression, post-translational modifications and protein aggregation could conceivably contribute to this neurotoxic effect, although a role of α-synuclein in MPTP-induced nigrostriatal degeneration remains to be demonstrated. Experiments in mice have attempted to address this issue but failed to produce a conclusive answer. Using α-synuclein null mice, an initial study showed that these animals were resistant to MPTP neurotoxicity, supporting a deleterious role of α-synuclein (51). A subsequent report, however, found that mice carrying a spontaneous deletion of the α-synuclein gene were not protected against MPTP and suggested that the genetic background of α-synuclein-deficient animals (rather than α-synuclein itself) determined their sensitivity to the toxicant (52). MPTP exposure causes significant α-synuclein changes in both mice and primates. A comparison of these changes between the two animal species reveals similarities but also important differences. For example, α-synuclein upregulation is a feature of both the mouse and primate MPTP models. The time-course of this effect is considerably more protracted in monkeys than in mice, however (11, 15). Moreover, previous work reported that no α-synuclein aggregates were formed in mice injected with MPTP (11, 53), whereas we document protein aggregation in the midbrain of MPTP-treated primates. These differences suggest that changes in α-synuclein caused by MPTP are more pronounced in primates than in rodents and raise the possibility that protein alterations may contribute to neuronal demise to a greater extent in the former.
In conclusion, we demonstrate the formation of nitrated, phosphorylated and aggregated α-synuclein as a consequence of MPTP-induced neuronal injury. Since these abnormal protein modifications are typical of human synucleinopathies, our observations support the possibility that toxic challenges play a role in the development of α-synuclein pathology in PD and other neurodegenerative diseases. Our data also suggest that specific modifications of α-synuclein (e.g. its phosphorylation and/or the formation of deleterious aggregates) could damage neurons and contribute to their demise in neurodegenerative processes. The findings suggest a pattern of evolution of MPTP-induced α-synuclein abnormalities that include the initial accumulation and aggregation of the protein at the axonal level and its retrograde buildup into dopaminergic cell bodies. Finally, the pronounced effects of MPTP on α-synuclein expression and modifications in the monkey model are consistent with the interpretation that the primate brain may be particularly vulnerable to pathological changes of α-synuclein triggered by toxic exposures.
The authors thank Dr. John P. Anderson (Elan Pharmaceuticals) for providing the antibody against phospho-Ser129 α-synuclein, Dr. John E. Duda (Philadelphia VA Medical Center) for discussion on pathological changes of α-synuclein and Ms. Martha Z. Isla for technical assistance.
Supported by Grants from the National Institutes of Health (ES12077) and the Backus Foundation.