Research reportLysosomal malfunction accompanies alpha-synuclein aggregation in a progressive mouse model of Parkinson’s disease
Introduction
Parkinson’s disease (PD) is a slow, progressive, neurodegenerative disorder involving the loss of dopaminergic neurons in the substantia nigra (SN), and functional deprivation of dopamine (DA) transmission in the striatum. Despite the typical locomotor impairment, clinical diagnosis of PD is not definitive and requires postmortem confirmation. The Lewy body (LB), a characteristic hallmark of the disease, is found in the brain stem and cortex as fibrous and punctate deposits or as an annulus with radially-oriented filaments surrounding a dense core [40]. These inclusion bodies typically contain cytoskeletal elements, α-synuclein, and other ubiquitinated protein aggregates [45]. They are also associated with accumulations of large secondary lysosomes containing lipofuscin, either embedded in the fibrous mass or at the periphery of the annulus [7], [16]. Lipofuscin contains peroxidized protein and lipid residues, and in the SN of humans, neuromelanin. These lysosomes normally accumulate in cells throughout the aging brain [20], [32], but accrue faster and in larger numbers under conditions of metabolic stress and in the presence of protease inhibitors [24], [44]. Alpha-synuclein has been identified in lipofuscin granules of brain stem neurons in PD [7]. Aggregation of this protein can injure cells, perhaps contributing to their demise [14], [36], [41], but it is unclear which factors contribute to its aggregation and whether the aggregates lead ultimately to cell death.
Investigations into inclusion formation and cell death in PD have been hampered by the lack of a truly chronic model to study the temporal pattern of progressive neurodegeneration and fully mimic PD symptomatology. It has been especially problematic to reveal the inclusion bodies. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced animal models, DA neurons in the SN become stressed in a manner believed to resemble that of PD itself, i.e. cellular levels of ATP drop and reactive oxygen species are formed [11]. The pyridinium ion (MPP+) of MPTP potently inhibits the mitochondrial complex I, but large doses administered over the short term seem to act on cells too rapidly for inclusions to form [52]. Nevertheless, aged non-human primates treated chronically with MPTP do show numerous inclusions that have LB-like features in the SN and cortex [15], [16].
It has been suggested that increased α-synuclein expression is required for inclusion formation. Certainly, conventional MPTP treatment significantly elevates α-synuclein gene expression [49]. However, studies of postmortem PD tissue reveal that α-synuclein mRNA is significantly decreased when compared to autopsy controls [33]. Furthermore, the intracellular clumps in neurons of transgenic, α-synuclein over-expressing mice are not typically LB-like [31], and the Lewy-like features in brain stem and motoneurons in mice expressing A53T mutant human α-synuclein are more representative of general α-synucleinopathy than idiopathic PD [48]. In the present study, we treated C57/bl mice chronically with MPTP, combined with probenecid (MPTP/p) to inhibit clearance of the toxin, to test whether with time, LB-like inclusions are generated in the brain and whether the formation of such inclusions requires elevations in α-synuclein gene expression.
Section snippets
Animals and treatment
Thirty-six male, C57/bl mice, 18–22 g, received 10 doses of MPTP (25 mg/kg in saline, s.c.) and probenecid (250 mg/kg in dimethyl sulfoxide [DMSO], i.p.) administered twice a week for 5 weeks. Animals treated with probenecid alone (n=36) formed the primary control groups each of which matched a MPTP/p groups in age and survival time. Two other control groups, one of which was left untreated (n=6) and the other administered vehicle (saline, n=6) were sacrificed 24 weeks after the initial 5-week
Results
The treatment of mice with MPTP/p results in significant cell death among SN neurons and a severe decline in dopaminergic function (Table 1). With tyrosine hydroxylase (TH) immunohistochemistry, few immunopositive neurons are present in the SN at 3 weeks after treatment (Fig. 1a, b). By 24 weeks post-MPTP/p, few TH-immunoreactive cell bodies remain, but there is a reappearance of TH-immunopositive processes (compare Fig. 1a, c). In control animals, no change in the pattern or numbers of
Discussion
In the present study, we demonstrate for the first time that granular and filamentous inclusions appear in the SN and cortex of a non-transgenic, rodent model of PD that has been treated chronically with MPTP (Table 1). The inclusions are α-synuclein- immunoreactive and develop in association with significantly reduced gene expression. They most closely resemble the aggregations of α-synuclein found in the cortex of PD patients and MPTP-treated squirrel monkeys [16], [40].
In this mouse model,
Acknowledgements
We are very grateful to S. Callen, S. Buzolich, J. Collins, E. Gates, and K. Schafbuch for technical assistance. We thank Drs. B. Wolozin and B. Roberts for their insightful comments on the manuscript. Supported by a NIH-NINDS grant NS41799 and a Wellcome Trust Biomedical Collaboration Grant.
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