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
). This transport is likely to be impaired by MPTP-induced injury of neuronal terminals (36
) 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
). Oxidative and nitrative damage also accompanies dopaminergic cell degeneration after MPTP exposure (40
) 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
). 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
). 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
). 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
). 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
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
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
). Moreover, previous work reported that no α-synuclein aggregates were formed in mice injected with MPTP (11
), 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.