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. 2005 Mar 1;102(9):3413-8.
doi: 10.1073/pnas.0409713102. Epub 2005 Feb 16.

Parkinson-like syndrome induced by continuous MPTP infusion: convergent roles of the ubiquitin-proteasome system and alpha-synuclein

Francesco Fornai  1 , Oliver M Schlüter, Paola Lenzi, Marco Gesi, Riccardo Ruffoli, Michela Ferrucci, Gloria Lazzeri, Carla L Busceti, Fabrizio Pontarelli, Giuseppe Battaglia, Antonio Pellegrini, Ferdinando Nicoletti, Stefano Ruggieri, Antonio Paparelli, Thomas C Südhof
Affiliations

Parkinson-like syndrome induced by continuous MPTP infusion: convergent roles of the ubiquitin-proteasome system and alpha-synuclein

Francesco Fornai et al. Proc Natl Acad Sci U S A. .

Abstract

In animals, sporadic injections of the mitochondrial toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) selectively damage dopaminergic neurons but do not fully reproduce the features of human Parkinson's disease. We have now developed a mouse Parkinson's disease model that is based on continuous MPTP administration with an osmotic minipump and mimics many features of the human disease. Although both sporadic and continuous MPTP administration led to severe striatal dopamine depletion and nigral cell loss, we find that only continuous administration of MPTP produced progressive behavioral changes and triggered formation of nigral inclusions immunoreactive for ubiquitin and alpha-synuclein. Moreover, only continuous MPTP infusions caused long-lasting activation of glucose uptake and inhibition of the ubiquitin-proteasome system. In mice lacking alpha-synuclein, continuous MPTP delivery still induced metabolic activation, but induction of behavioral symptoms and neuronal cell death were almost completely alleviated. Furthermore, the inhibition of the ubiquitinproteasome system and the production of inclusion bodies were reduced. These data suggest that continuous low-level exposure of mice to MPTP causes a Parkinson-like syndrome in an alpha-synuclein-dependent manner.

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Figures

Fig. 1.
Fig. 1.
MPTP-induced activation of [14C]2-DG uptake. (a–d) False-color autoradiographs of brain sections from mice 1 h after injection with [14C]2-DG. 2-DG uptake was monitored in control mice (a), mice that were analyzed 1 h (b) or 7 days (c) after injections with a single dose of MPTP (30 mg/kg), and mice that were analyzed after 28 days of continuous MPTP infusion (30 mg/kg daily). (e–j) 2-DG uptake was quantified by film densitometry (n = 8–10 mice per group) in the neostriatum (e), globus pallidus (f), thalamus (g), frontal cortex (h), substantia nigra pars compacta (i), and substantia nigra pars reticulata (j). Asterisks indicate that the treated sample is significantly different (P < 0.05; ANOVA) from the controls (single asterisk) or from both the control and the MPTP-bolus injected sample 7 days after the injection (double asterisk). All data shown in this in all subsequent figures represent means ± SEMs.
Fig. 2.
Fig. 2.
Striatal proteasome activity after MPTP treatments. (a) Relative chymotrypsin-like, trypsin-like, and peptidyl-glutamyl-peptide hydrolyzing (PGPH) proteasome activities in mice exposed to a single (30 mg/kg) or four separate doses (20 mg/kg each) of MPTP (n = 5 mice at each time interval). Asterisks indicate statistically significant differences (P < 0.05) from baseline proteasome activity (single asterisk) or from both baseline proteasome activity and activity after single MPTP dose (double asterisk). (b) Delayed and prolonged inhibition of proteasome activity after continuous MPTP administration (1, 5, or 30 mg/kg MPTP daily) for the indicated time periods. Asterisks indicate statistically significant differences (P < 0.05) from baseline proteasome activity (single asterisk) or from both baseline proteasome activity and activity after lower MPTP doses (1 and 5 mg/kg, daily, double asterisk; n = 5 mice).
Fig. 3.
Fig. 3.
Neurotoxicity induced by continuous MPTP administration. (a) Representative tyrosine hydroxylase (TH)-stained sections of the substantia nigra from mice that were continuously treated for 28 days with control pump infusions or with infusions of 1, 5, or 30 mg MPTP/kg daily. (Scale bar, 600 μm.) (b and c) TH-positive cell counts in the substantia nigra (b) and semiquantitative densitometric measurements of the TH signal in striatum (c)(n = 10 mice per group). (d) Striatal monoamine levels in MPTP-treated mice (n = 10 mice per group). Asterisks indicate statistically significant differences (P < 0.05) of a sample compared to control (single asterisks) or to both the control and the lower MPTP dose (double asterisks).
Fig. 4.
Fig. 4.
Continuous MPTP administration induces neuronal inclusions and a dopamine-dependent behavioral phenotype. (a and b) Inclusion bodies in the substantia nigra from mice treated for 28 days with continuous MPTP infusions (30 mg/kg daily). Inclusions were stained for ubiquitin (a) and α-synuclein (b). (Scale bars, 120 μm at Left and 35 μm at Right.) (c and d) Electron micrographs of inclusions observed 72 h (c) or 30 days (d) of MPTP infusion. (Scale bars, 0.1 μm.) Note that similar inclusions were observed in the locus coeruleus (Fig. 12). (e and f) Open field behaviors of mice infused continuously with control solution or intermediate (5 mg/kg daily) and high doses of MPTP (30 mg/kg daily). Suppression of open field activity (e) and rearing (f) by continuous MPTP administration was reversed by the mixed dopamine agonist apomorphine (APO). Asterisks indicate statistically significant differences from control mice (see Movies 1–3, which are published as supporting information on the PNAS web site).
Fig. 5.
Fig. 5.
Effect of an α-synuclein deletion on MPTP toxicity. (a) Spontaneous horizontal activity and rearing of control and MPTP-treated wildt-ype and α-synuclein KO mice monitored in the open field test (30 mg of MPTP per kg daily for 28 days; n = 8). (b) Uptake of [14C]2-DG in littermate wild-type and α-synuclein KO mice that were continuously infused for 7 days with control or MPTP (30 mg/kg daily) solution. Pictures display false-color autoradiograms. (c) Proteasome activity in control and α-synuclein KO mice continuously infused with MPTP (30 mg per kg of body weight daily). Proteasome activities in the substantia nigra are depicted as percent of control (means ± SEMs) as a function of time after beginning of the infusions (five mice per group). In a and c, asterisks indicate statistically significantly different values (P < 0.05) from controls.
Fig. 6.
Fig. 6.
Deletion of α-synuclein alleviates neurodegeneration caused by continuous MPTP infusion. (a) Representative TH-labeled sections of the striatum from wild-type and α-synuclein KO mice treated with control infusions or 30 mg MPTP/kg (scale bars = 2 mm). (b) Semiquantitative measurements of the TH immunoreactivity in the striatum by densitometry of light microscopy pictures in control and MPTP-treated wild-type and α-synuclein KO mice (n = 10 mice). (c) Quantitation of TH-positive cells in the substantia nigra from littermate wild-type and α-synuclein KO mice that were treated with either control or MPTP infusions (30 mg/kg daily; n = 5). (d) Electron microscopic quantitation of cells with “whorl” inclusion bodies (see Fig. 4c)in the substantia nigra of littermate wild-type and α-synuclein KO mice that were treated with either control or MPTP infusions (30 mg/kg daily; n = 5 mice). (e) Quantitation of striatal levels of dopamine, its metabolite DOPAC, and serotonin in wild-type and α-synuclein KO mice that were treated with control infusions or 30 mg of MPTP per kg of body weight daily (n = 10). Asterisks indicate that the respective value for the wild-type mice is statistically significantly different (P < 0.05) from that obtained for the other three conditions (control-treated wild-type and α-synuclein KO and MPTP-treated α-synuclein KO mice). MPTP-treated α-synuclein KO mice exhibit a statistically significant difference with control treated mice only for whorls (d), but even here the number of whorls in the KO mice is significantly less than in wild-type mice (indicated by a double asterisk).
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