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. 2008 Nov 25;105(47):18578-83.
doi: 10.1073/pnas.0804373105. Epub 2008 Nov 14.

Divalent metal transporter 1 (DMT1) contributes to neurodegeneration in animal models of Parkinson's disease

Julio Salazar  1 , Natalia Mena, Stephane Hunot, Annick Prigent, Daniel Alvarez-Fischer, Miguel Arredondo, Charles Duyckaerts, Veronique Sazdovitch, Lin Zhao, Laura M Garrick, Marco T Nuñez, Michael D Garrick, Rita Raisman-Vozari, Etienne C Hirsch
Affiliations

Divalent metal transporter 1 (DMT1) contributes to neurodegeneration in animal models of Parkinson's disease

Julio Salazar et al. Proc Natl Acad Sci U S A. .

Abstract

Dopaminergic cell death in the substantia nigra (SN) is central to Parkinson's disease (PD), but the neurodegenerative mechanisms have not been completely elucidated. Iron accumulation in dopaminergic and glial cells in the SN of PD patients may contribute to the generation of oxidative stress, protein aggregation, and neuronal death. The mechanisms involved in iron accumulation also remain unclear. Here, we describe an increase in the expression of an isoform of the divalent metal transporter 1 (DMT1/Nramp2/Slc11a2) in the SN of PD patients. Using the PD animal model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication in mice, we showed that DMT1 expression increases in the ventral mesencephalon of intoxicated animals, concomitant with iron accumulation, oxidative stress, and dopaminergic cell loss. In addition, we report that a mutation in DMT1 that impairs iron transport protects rodents against parkinsonism-inducing neurotoxins MPTP and 6-hydroxydopamine. This study supports a critical role for DMT1 in iron-mediated neurodegeneration in PD.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
DMT1 expression in human mesencephalon. (A) DMT1 expression in neuromelanin (brown)-containing neurons of control subjects (images representative of 6 subjects studied). Neuromelanin-positive neurons from SNpc exhibited moderate Pan-DMT1 immunoreactivity (dark gray; Upper), whereas those from VTA (Lower) had scarce immunostaining. (B) DMT1 expression in SNpc neuromelanin-positive neurons in a PD subject (images representative of 7 patients studied). Pan-DMT1 immunostaining in neuromelanin-positive neurons (Top) was comparable to control. Pan-DMT1 was found in neuromelanin-positive neurons with and without Lewy bodies (arrows). PD patients also exhibited abundant labeling in small cells (<10 μm; arrowheads) in the ventral aspects of the SNpc. DMT1 +IRE immunolabeling (Middle and Bottom Right) localized like Pan-DMT1 immunoreactivity. The presence of iron accumulation in neuromelanin-positive neurons (arrows) and glial cells (arrowheads) in subjects studied was confirmed by Perls staining (blue; Bottom). (C) Colocalization of microglial marker CD68 (green) and Pan-DMT1 (magenta). (Scale bar and Inset width: A and B, 60 μm; C, 5 μm.) (D) DMT1 +IRE and −IRE immunoblots using extract of SNpc of controls and PD patients. Deglycosylation control using peptide N-glycosidase F (PNGase) (first lane of immunoblot). Bar charts quantify iron content and DMT1 blots normalized by actin in controls (black; n = 7) and PD patients (white; n = 7). **, P < 0.01.
Fig. 2.
Fig. 2.
DMT1 expression in the mesencephalon of MPTP-intoxicated mice. (A) Western blot analysis of DMT1 C-terminal isoforms and actin expression in ventral mesencephalon homogenates of control and MPTP-treated mice (n = 4 or 5 per day). (B) Time course of DMT1 +IRE and DMT1 −IRE expression changes (by immunoblot quantification; Top and Middle) and percentage of dopaminergic cell loss (by stereological cell counting of TH-positive DNs; Bottom) after acute MPTP intoxication. **, P < 0.01 compared with control; ##, P < 0.05 compared with day 1. (C and D) Localization of DMT1 isoforms and cell type-specific markers in the coronal mesencephalic sections of control and MPTP-treated mice. (C, Top) Modest DMT1 +IRE expression in the SNpc of control mice. (C, Middle and Bottom) DMT1 +IRE expression (red) in TH-positive dopaminergic neurons (green) and CD11b+ activated microglia (cyan) 2 days after MPTP intoxication. (D, Upper) DMT1 −IRE expression (red) in TH-positive dopaminergic neurons (green) in the SNpc of control mice. (D, Lower) DMT1 −IRE expression (red) in TH-positive dopaminergic neurons (green) in SNpc 2 days after MPTP intoxication. (Scale bar and inset width: 100 μm.)
Fig. 3.
Fig. 3.
MPTP-induced dopaminergic cell loss in +/+, +/mk, and mk/mk mice. (A) Peroxidase/DAB immunohistochemistry for TH on coronal mesencephalic sections from saline-injected and MPTP-intoxicated +/+ (WT) and mk/mk mice. The SNpc is delineated. (Scale bar: 100 μm.) (B) Bar graph of stereological counts of TH-positive cells in the SNpc of saline- and MPTP-injected mice (7 days after MPTP intoxication). **, P < 0.01.
Fig. 4.
Fig. 4.
Amphetamine-induced rotational behavior and dopaminergic cell loss in +/+, +/b, and b/b rats after 6-OHDA intrastriatal injection. (A) Bar graph of amphetamine-induced rotational behavior 7 days after 6-OHDA injection in wild-type Fischer 344 (+/+), heterozygous (+/b), and Belgrade (b/b) rats. (B) Bar graph of stereological nigral TH-positive DN counts in sham-injected and 6-OHDA-injected rats 14 days after injection. **, P < 0.01. (C) Representative peroxidase/DAB immunohistochemistry for TH on coronal mesencephalic sections +/b (control) and b/b (Belgrade) rats 14 days after 6-OHDA injection. (Scale bar: 300 μm.) (Insets) Enlargements of the outlined areas.
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