α-Syn is a 14 kDa, soluble, naturally unfolded protein, mainly present in the presynaptic terminal of the neurons. The cytoplasmic accumulation of misfolded α-syn as LB forms the pathological hallmark of both sporadic and familial PD [26]. Point mutations as well as duplication and triplication of the α-syn gene, SNCA, have been reported in PD patients of several families. The physiological function of α-syn is not fully understood. However, it has been shown that the germ line deletion of α-syn in mice impairs neurotransmitter release. Moreover, α-syn deficient mice are resistant to the mitochondrial neurotoxin, MPTP, while mice presenting the ectopic expression of α-syn are sensitive to MPTP. siRNA mediates the knockdown of α-syn in mice, thereby making them resistant to MPTP toxicity [18, 27]. The above observation suggests that the oxidative stress generated in the mitochondria due to MPTP injection augments the pathological nature of α-syn in producing toxic species. However, the data showing α-syn aggregation in mice injected with MPTP are conflicting [28–30]. It has been reported that the exposure of mice to MPTP does not cause any LB-like pathology, although profound DA neurodegeneration has been observed [30]. One report demonstrated the increased expression of syn mRNA in mice that received prolonged MPTP treatment [29]. Moreover, Meredith et al. [28] provided evidence that MPTP injection causes the accumulation of granular and filamentous α-syn inclusions in DA neurons. However, other researchers negated the claim and reported that MPTP causes neurodegeneration without α-syn inclusion [30]. It is noteworthy to mention that different doses of MPTP have been used in mice models of acute, sub-chronic, and chronic PD. Interestingly, only a chronic dose of MPTP has the ability to increase the expression of α-syn mRNA; an acute dose does not. Although only monkeys injected with MPTP have shown intraneuronal inclusions suggestive of LBs [31, 32], the major drawback of this mouse model was the lack of α-syn inclusion in MPTP treated mice. Moreover, in monkeys, a low dose of MPTP has been shown to cause a greater loss of DA nerve terminals in the putamen than in the caudate nucleus [33, 34]. Thus, one of the arguments raised is that this discrepancy in α-syn inclusion generation is a result of the animal species used, the dosage, and duration of MPTP administration in these models.
Growing number of experimental data suggest that the inoculation of a pathological form of α-syn, such as PFF, in mice brain, can induce the progressive propagation and spread of endogenous α-syn throughout the interconnected brain regions, through cell-to-cell transmission mechanisms [4, 5, 35, 36]. However, this α-syn propagation requires the presence of endogenous α-syn, A previous study reported that α-syn knockout mice failed to show any such spread and aggregation [4]. However, transgenic mice expressing WT or mutant α-syn did not self-initiate the propagation, thereby indicating that PFF injection, which acts as a seeding material, is a prerequisite. Thus, the α-syn fibrils mediating the seeding process are very critical for the initiation and propagation of endogenous α-syn. It is noteworthy to mention that α-syn propagation has only been observed using a pS129 α-syn-specific antibody. Therefore, the hyper-phosphorylation of α-syn at serine 129 residue is considered to be a pathological consequence which induced by α-syn PFF and plays a critical role in the aggregation of α-syn, and in PD progression [5]. Previous reports have shown that the minimum time required to observe the spread of α-syn in the SNpc area is at least a month or more after the inoculation of α-syn PFF at multiple sites. However, DA neuronal death does not appear before three months post the injection of PFF.
In the current study, we examined to see if the injection of different doses of MPTP (one low and one high dose) in mice pre-inoculated with α-syn PFF had any effect on the spread, toxicity, and pathology of endogenous α-syn. To accomplish this, we administered a single stereotaxic injection of α-syn PFF in the striatum of mice, and challenged them with two different doses of MPTP: a high dose of 25 mg/kg.b.wt and a low dose of 10 mg/kg.b.wt. A high dose of MPTP has been known to cause the rapid destruction of DA neurons in the nigrostriatal pathway, with concomitant loss of DAT, and has been commonly used in PD mouse models. A low dose of MPTP has been reported to induce mild effects in DA neurons without significant destruction of these neurons [20]. MPTP, following conversion to MPP+, inhibits complex 1 of the mitochondrial respiratory chain complex and selectively generates reactive oxygen species (ROS) in the DA neurons [37]. We observed that the exposure of PFF inoculated mice to a low dose of MPTP significantly increased the spread and accumulation of endogenous α-syn, from the striatum to the SNpc, as detected by pS129 α-syn antibody. Surprisingly, we found that the exposure of PFF inoculated mice to a high dose of MPTP reduced the spread of α-syn from the striatum to the SNpc, and the localization of α-syn in the cytoplasm. An in vitro study demonstrated that α-syn, owing to its chemical structure, had the tendency to become less soluble and form insoluble, high molecular weight aggregates when exposed to an oxidizing agent [38]. MPTP can generate reactive oxygen species (ROS) and increase the amount of insoluble α-syn aggregates. Thus, MPTP intensifies this aggregation process [39] and results in more detrimental effects on neurons. In our current study, we observed a higher DA neuronal death in PFF injected mice exposed to a high dose of MPTP, leading to the lower availability of healthy DA neurons for the active spread of α-syn.
A growing body of evidence has shown that the proteinase K-resistant α-syn species formed in the brain of human PD patients have the characteristics of intracytoplasmic inclusions of LBs [40]. To examine this phenomenon, prior to immunostaining, we treated the brain section with proteinase K to detect proteinase K-resistant aggregates of α-syn using a conformation-specific antibody, as reported previously [41]. In addition to removing the soluble synuclein, proteinase K has also been extensively used to retrieve the antigen [42, 43]. In α-syn PFF injected mice with or without MPTP challenge, we found that the proteinase K-resistant α-syn were accumulated in the SNpc area of the brain. However, in α-syn injected mice challenged with a low dose of MPTP, we found an abundant amount of proteinase K-resistant α-syn species. In addition, we did not observe α-syn positive staining in the SNpc region of control mice or mice treated with MPTP alone following proteinase-k treatment. Surprisingly, we observed significant nuclear localization of α-syn in α-syn PFF injected animals challenged with a high dose of MPTP.
In transgenic fruit flies and tissue culture systems, it has been reported that α-syn is translocated to the nucleus, where it promotes cell death by inhibiting histone acetylation. However, the administration of histone deacetylase inhibitor was found to rescue the cells from α-syn toxicity [44]. Recently, it has been demonstrated that TRIM28 regulates the nuclear accumulation and toxicity of α-syn [45]. Transgenic mice expressing human α-syn have been reported to show nuclear translocation with distinct cytoplasmic inclusion [46]. Another study revealed that α-syn promotes cell death by activating nitric oxide synthase (NOS), which leads to the damage of DNA and activation of polyadenosine 5'-diphosphate-ribose polymerase-1 (PARP-1), leading to parthanatos [47]. In tissue culture, it has been found that the oxidative stress induced by H2O2 increases the nuclear translocation of α-syn, leading to increased cell death [48]. In our current study, we observed that the exposure of PFF inoculated mice to a high dose of MPTP led to the nuclear translocation of proteinase K-resistant α-syn, and the highest degree of DA neuronal death. Our results are in agreement with the aforementioned findings, and support further that α-syn mediates the pathological effect leading to neurodegradation by increasing its nuclear translocation rather than spread.
Neuroinflammation significantly contributes to neurodegeneration not only in animal models of PD [49] but also in PD patients [50]. α-Syn aggregation elicits microglial cell and astrocyte response, which mediate neuroinflammation by secreting inflammatory mediators such as reactive oxygen species, cytokines, and chemokines that eventually contribute to enhanced DA neuronal death in the SNpc area [51, 52]. Mice over-expressing human α-syn show an increased level of activated microglial cells and TNF-α in the striatum and SNpc area [53]. It has been reported that extracellular α-syn released from the neuron elicits microglial inflammatory response by acting as an endogenous agonist for toll-like receptor 2 (TLR2) [54]. In mice, MPTP induces glial cell response by activating microglial cells and astrocytes. In MPTP treated mice, activated microglial cells have a large cell body with short and thick processes while activated astrocytes show a large cell body with long and thick processes. Consistent with the above report, in mice that received a high dose of MPTP along with α-syn PFF, we found a significant increase in the number of activated astrocytes and microglial cells in the SNpc area. In the same group of animals, we also observed the highest DA neurons loss with nuclear translocation of α-syn within the TH neurons. The prior hypothesis of α-syn stated that α-syn could be taken up by neurons, where it tends to act as a seed and enhance the aggregation of endogenous α-syn and release it into the extracellular space. These α-syn aggregates then enter the neighboring neurons, microglial cells or astrocytes [55–57]. α-Syn, upon endocytosis by astrocytes, causes a change in gene expression, leading to an increase in the induction of pro-inflammatory cytokines and chemokines which lead to an inflammatory response which mediates the pathological effect [57]. Compared to the control, in α-syn injected mice, we observed an increase in the number of activated astrocytes in the SNpc area. This activation became higher when we combined α-syn PFF with a high dose of MPTP, a mitochondrial complex I inhibitor that plays a key role in the massive release of free radicals following its intraperitoneal administration in rodents. Collectively, aggregated α-syn and a high dose of MPTP have the ability to enhance the activation of glial cells and amplify the cascade of nigrostriatal degeneration.
Finally, we performed behavioral studies on our experimental mice. In PFF inoculated mice challenged with MPTP, in addition to the effect on the neurons in the SNpc, we also found compromised behavior, as measured by the rotarod test. However, we did not observe any significant change in the grip test performance of the experimental animals compared to the PBS injected and saline treated control animals. In summary, through this study, we have demonstrated the significant spread of α-syn and the moderate DA neuronal loss in mice injected with a low dose of MPTP in conjunction with α-syn PFF. Thus, the administration of a low dose of MPTP to PFF inoculated mice enables the use of these animal models in future mechanistic studies and for novel drug development.