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Pathologic Modifications of α-Synuclein in 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP)-Treated Squirrel Monkeys

Alison L. McCormack PhD, Sally K. Mak PhD, Maryam Shenasa BS, William J. Langston MD, Lysia S. Forno MD, Donato A. Di Monte MD
DOI: http://dx.doi.org/10.1097/NEN.0b013e318180f0bd 793-802 First published online: 1 August 2008

Abstract

α-Synuclein expression is increased in dopaminergic neurons challenged by toxic insults. Here, we assessed whether this upregulation is accompanied by pathologic 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 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. After toxicant exposure, α-synuclein deposition occurred at the level of neuronal axons in which amorphous protein aggregates were observed by immunoelectron microscopy. In a subset of these axons, immunoreactivity for α-synuclein was still evident after tissue digestion with proteinase K, further indicating the accumulation of insoluble protein. These data indicate that toxic injury can induce 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.

Key Words
  • Monkey
  • MPTP
  • Nitration
  • Parkinson
  • Phosphorylation
  • Substantia nigra
  • Synuclein

Introduction

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, such as dementia with Lewy bodies and multiple system atrophy (4, 5). Most patients affected by these diseases do not carry any α-synuclein mutations, indicating that toxic properties inherent to normal (i.e. nonmutated) α-synuclein are likely to underlie its role in disease pathogenesis (6-8). Furthermore, deleterious effects of normal α-synuclein are clearly supported by the relationship between SNCA genomic multiplication, enhanced expression of the wild-type protein, and the development of familial parkinsonism (9, 10). This relationship is of particular relevance because it suggests that any condition capable of augmenting α-synuclein levels may contribute to the pathogenesis of PD and other synucleinopathies.

Vila et al (11) were the first to report an intriguing link between toxicant-induced neuronal damage and α-synuclein upregulation in an animal model of PD. After 5 consecutive daily injections of 1-methyl-4-phenyl-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 association between toxicant exposure and α-synuclein alterations is further supported by studies in nonhuman primates. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration was found to cause a sustained (i.e. at least 1 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 et al (16) proposed that this latter effect may model the early stages of Lewy body formation. 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.

Materials and Methods

Animals and Treatment

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.

Tissue Preparation

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 2 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).

Immunohistochemistry

One set of sections was immunostained for bright-field microscopy. After washing in 0.01 mol/L phosphate-buffered saline (PBS; pH 7.4), 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 (1:4000; Millipore, Billerica, MA), mouse anti-phospho-Ser 129 α-synuclein (1:3000; gift from Elan Pharmaceuticals, South San Francisco, CA), sheep anti-β-synuclein (1:600; Chemicon, Temecula, CA), or mouse anti-synaptophysin (1:1000; Chemicon). Immunolabeling was visualized using the avidin-biotin peroxidase complex method with 3,3′-diaminobenzidine 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 previously described (19).

Proteinase K pretreatment 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 previously described. 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-glial fibrillary acidic protein (GFAP) (1:600; Dako, Carpinteria, CA), rabbit anti-ionizing calcium-binding adaptor molecule 1 (1:1000; Biocare Medical, Concord, CA), mouse anti-nitrated α-synuclein (1:4000; Millipore), or mouse anti-phospho-Ser 129 α-synuclein (1:3000; gift from Elan Pharmaceuticals). Sections were then incubated with the appropriate fluorescent secondary antibody conjugated to either fluorescein isothiocyanate or Cy-3 (1:500; Jackson ImmunoResearch Laboratories, West Grove, PA). Tissues immunostained for phospho-Ser 129 α-synuclein were pretreated in formic acid as previously described. 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 immunoglobulin G were devoid of staining.

Immunoelectron Microscopy

Tissues were embedded in LR White resin, and thin sections were 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 postfixed 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.

Quantification of α-Synuclein-Positive Dystrophic Axons

Tissues from 3 control monkeys and 3 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).

Results

MPTP-Induced Accumulation of α-Synuclein Within Dopaminergic Cell Bodies

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 an 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 1 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 3 proteins is two-fold. 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 (Figs. 1A, C, E). In MPTP-treated animals, neuromelanin-containing cells (i.e. dopaminergic neurons) displayed robust α-synuclein immunoreactivity (Fig. 1B). However, MPTP treatment did not enhance β-synuclein or synaptophysin immunostaining, nor did it change their distribution between synaptic profiles and neuronal cell bodies (Figs. 1D, F).

FIGURE 1.

α-Synuclein, but not other synaptic proteins, is accumulated within nigral dopaminergic cell bodies of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys. Squirrel monkeys were given a single subcutaneous injection of either saline or MPTP and killed 4 weeks later. Representative midbrain sections from control (A, C, E) and MPTP-treated (B, D, F) animals were immunostained for α-synuclein (A, B), β-synuclein (C, D), or synaptophysin (E, F), and counterstained with cresyl violet (purple). Increased immunoreactivity (brown) for α-synuclein, but not β-synuclein or synaptophysin, is evident within neuromelanin-loaded (black granules) nigral dopaminergic cell bodies in the MPTP-treated monkey. Scale bar = (F) 10 μm (A-F).

Axonal α-Synuclein Pathology as a Consequence of MPTP Exposure

In addition to increased α-synuclein immunoreactivity within neuronal cell bodies, numerous α-synuclein-positive dystrophic axons were consistently observed in the ventral midbrain of MPTP-treated, but not control monkeys (Figs. 2A, B). Swollen axons varied in size (up to 20 μm in diameter) and morphology, resembling features similar to α-synuclein-positive neurites in PD midbrain (Figs. 2C, D). They were not evenly distributed across the region but were more concentrated along the dorsomedial tier of the substantia nigra and the ventral portion of the ventral tegmental area. This pattern most likely reflects the topographic distribution of the ascending projections of nigral dopaminergic neurons (24). The specificity of axonal α-synuclein pathology was indicated by lack of axonal β-synuclein or synaptophysin immunoreactivity (data not shown).

FIGURE 2.

Axonal accumulation of α-synuclein in the midbrain of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys resembles pathologic changes in Parkinson disease (PD) patients. (A-C) Representative sections of the ventral midbrain from monkeys injected once with either saline (A) or MPTP (B, C) and killed 4 weeks later. Tissues were immunostained with an antibody against α-synuclein and counterstained with cresyl violet. Numerous α-synuclein-immunoreactive (brown) axons are shown at low (B) and high (C) magnification in the ventral midbrain of the MPTP-treated animal. (D; panel and inset) Pathologic axons in the ventral midbrain of a PD patient. A representative midbrain section was immunostained with an antibody against α-synuclein and counterstained with cresyl violet. Scale bar = (B) 100 μm (A, B). Scale bars = (C, D, and inset) 20 μm.

Neuritic accumulation of α-synuclein was confirmed by immunoelectron microscopy (IEM). α-Synuclein labeling was increased within myelinated and unmyelinated neurites (axons and dendrites) in MPTP- (Fig. 3B) compared with 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. After MPTP administration, enlarged myelinated axons labeled with α-synuclein were frequently present in IEM sections (Figs. 3D, E); these most likely correspond to the dystrophic axons observed by light microscopy.

FIGURE 3.

Immunoelectron microscopy (IEM) images of α-synuclein accumulation within dystrophic neurites. Tissue punches of the ventral midbrain of monkeys treated with saline or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were processed for IEM with an anti-α-synuclein antibody. (A, B) Representative electron micrographs show increased α-synuclein immunolabeling (black particles) within myelinated (B) and unmyelinated (B; inset) neurites in the MPTP-treated monkey compared with the control (A). (C, D) Representative electron micrographs show normal tissue architecture in a control (C) and an enlarged dystrophic axon in an MPTP-injected animal (D; arrow). (E) At higher magnification, accumulation of α-synuclein (black particles) is more evident within a dystrophic axon in an MPTP-treated monkey. Scale bar = (B) 1 μm (A, B). Scale bar = (inset) 1 μm. Scale bar = (D) 4 μm (C, D). Scale bar = (E) 1 μm.

Lack of α-Synuclein Accumulation Within Glial Cells

Accumulation of α-synuclein within nonneuronal 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). Because 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 (ionizing calcium-binding adaptor molecule 1) 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 distribution of α-synuclein compared with GFAP and ionizing calcium-binding adaptor molecule 1 immunoreactivities, with no evidence of α-synuclein accumulation within astrocytes or microglia (Fig. 4).

FIGURE 4.

α-Synuclein does not colocalize with astrocytic or microglial markers. (A-C) A representative section of the ventral midbrain from a monkey injected once with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and killed 4 weeks later was colabeled with anti-α-synuclein (A) and anti-GFAP (an astrocytic marker; B). The arrow in (A) indicates an enlarged dystrophic axon positively stained for α-synuclein. The merged image (C) shows lack of α-synuclein immunoreactivity within astrocytes. (D-F) A representative section of the ventral midbrain from an MPTP-treated monkey was colabeled with anti-α-synuclein (D) and anti-ionizing calcium-binding adaptor molecule 1 (a microglial marker; E). The arrow in (D) indicates an enlarged α-synuclein-immunoreactive axon. The merged image (F) shows lack of α-synuclein and ionizing calcium-binding adaptor molecule 1 colocalization. Scale bar = (F) 10 μm (A-F).

MPTP-Induced Nitration and Phosphorylation of α-Synuclein

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 (Figs. 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 (Figs. 5D-F) that were immunoreactive for unmodified α-synuclein were also labeled for the nitrated form of the protein.

FIGURE 5.

Nitrated α-synuclein within nigral dopaminergic cell bodies and dystrophic axons of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MFTP)-treated monkeys. Squirrel monkeys received a single subcutaneous injection of either saline or MPTP and were killed 4 weeks later. (A-C) Representative midbrain sections from a control (A) and an MPTP-treated (B, C) animal were immunostained with anti-nitrated α-synuclein and counterstained with cresyl violet (purple). Immunoreactivity for nitrated α-synuclein (brown) is shown within a neuromelanin-containing nigral neuron (B) and dystrophic neurites (C) after MPTP treatment. (D-F) A representative midbrain section from an MPTP-treated animal was processed for confocal microscopy. Tissue was dual-labeled with anti-α-synuclein (D) and anti-nitrated α-synuclein (E) antibodies. The merged image (F) shows that α-synuclein is nitrated in some, but not all, dystrophic neurites. Scale bar = (B) 10 μm (A, B). Scale bar = (C) 10 μm. Scale bar = (F) 10 μm (D-F).

Another posttranslational 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 (Figs. 6A-C). Phospho-Ser 129 α-synuclein was also present within some, but not all, dystrophic axons. These neurites could be double labeled using antibodies against unmodified α-synuclein and phospho-Ser 129 α-synuclein (Figs. 6D-F).

FIGURE 6.

Phosphorylated α-synuclein within nigral dopaminergic cell bodies and axonal profiles of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-exposed monkeys. Squirrel monkeys received a single subcutaneous injection of either saline or MPTP and were killed 4 weeks later. (A-C) Representative midbrain sections from a control (A) and an MPTP-treated (B, C) animal were immunostained with an anti-phospho-Ser 129 α-synuclein antibody and counterstained with cresyl violet (purple). Immunoreactivity for phospho-Ser 129 α-synuclein (brown) is shown within a neuromelanin-containing nigral neuron (B) and dystrophic neurites (C) after MPTP treatment. (D-F) A representative midbrain section from an MPTP-treated animal was processed for confocal microscopy. Tissue was dual-labeled with anti-α-synuclein (D) and anti-phospho-Ser 129 α-synuclein (E) antibodies. The merged image (F) shows that α-synuclein is phosphorylated at Ser129 in some but not all dystrophic neurites. Scale bar = (B) 10 μm (A, B). Scale bar = (C) 10 μm. Scale bar = (F) 10 μm (D-F).

α-Synuclein Aggregation Within MPTP-Injured Axons

Immunoelectron microscopy observations provided initial evidence of α-synuclein deposition within neurites of MPTP-challenged monkeys (see previous sentences; Fig. 3B). Further evaluation of protein aggregation was performed 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 (Figs. 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 (Figs. 7C, D). In contrast, treatment with proteinase K reduced but did not completely eliminate α-synuclein immunoreactivity associated with some dystrophic axons (Figs. 7E, F), suggesting the presence of both soluble and insoluble protein.

FIGURE 7.

Proteinase K-resistant axonal pathology in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-exposed monkeys. Squirrel monkeys received a single subcutaneous injection of either saline or MPTP and were killed 4 weeks later. Representative midbrain sections from a saline- (A, B) and an MPTP-treated (C-F) animal were incubated in either buffer (A, C, E) or proteinase K (B, D, F). Sections were subsequently immunostained with an antibody against α-synuclein (brown) and counterstained with cresyl violet (purple). The robust immunoreactivity observed within nigral cell bodies of the MPTP-exposed animal (C) was removed by proteinase K digestion, leaving only neuromelanin granules (black) (D). A subset of dystrophic axons in the MPTP-treated monkey retained α-synuclein immunoreactivity even after proteinase K treatment (F). Scale bar = (D) 10 μm (A-D). Scale bar = (F) 10 μm (E, F).

The number of enlarged α-synuclein-labeled axons was counted in 2 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, after toxicant exposure, aggregated protein was present in approximately 15% of α-synuclein-immunoreactive dystrophic axons.

Discussion

Administration of MPTP to nonhuman 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 (15) reported increased levels of α-synuclein mRNA and protein in squirrel monkeys at 1 week and 1 month after a single injection of MPTP. 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 et al (16) also found a pronounced redistribution of α-synuclein that seemed to cluster within neuronal somata in the form of α-synuclein-immunoreactive granules 10 days after the initial MPTP administration. 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 because no α-synuclein immunoreactivity labeled astrocytes or microglia. Our data also indicate that increased expression is relatively selective for α-synuclein because 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. 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 succeeding sentences). Thus, the combination of protein upregulation, toxic lesion of the terminals, and obstructive deposition could lead 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). Because 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 accompany dopaminergic cell degeneration after MPTP exposure (40-42); 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). Relatively more clear, however, are the pathologic implications of another posttranslational modification of α-synuclein, i.e. phosphorylation at Ser129. 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 kinases 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, for example enhancing its tendency to aggregate, 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 because the presence of phosphorylated α-synuclein (a) could be a key event in the development of further α-synuclein pathology (19) and (b) 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 et al (49) have described the formation of eosinophilic Lewy body-like inclusions in squirrel monkeys injected with MPTP. This observation seems to be at odds with results of the current study in which we failed to identify organized Lewy body-like structures in MPTP-treated nonhuman 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 et al (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 postinjury, 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 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, posttranslational 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). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine exposure causes significant α-synuclein changes in both mice and primates. A comparison of these changes between the 2 animal species reveals similarities but also important differences. For example, α-synuclein upregulation is a feature of both the mouse and primate MPTP models. However, the time course of this effect is considerably more protracted in monkeys than in mice (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 than the latter model.

In conclusion, we demonstrate the formation of nitrated, phosphorylated, and aggregated α-synuclein as a consequence of MPTP-induced neuronal injury. Because 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 pathologic changes of α-synuclein triggered by toxic exposures.

Acknowledgments

The authors thank John P. Anderson, PhD (Elan Pharmaceuticals) for providing the antibody against phospho-Ser 129 α-synuclein; John E. Duda, MD (Philadelphia VA Medical Center), for discussion on pathologic changes of α-synuclein; and Martha Z. Isla, for technical assistance.

Footnotes

  • Supported by Grant No. ES12077 from the National Institutes of Health and the Backus Foundation.

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