NADPH oxidase mediates oxidative stress in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease - PubMed (original) (raw)

NADPH oxidase mediates oxidative stress in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease

Du-Chu Wu et al. Proc Natl Acad Sci U S A. 2003.

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder of uncertain pathogenesis characterized by a loss of substantia nigra pars compacta (SNpc) dopaminergic (DA) neurons, and can be modeled by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Both inflammatory processes and oxidative stress may contribute to MPTP- and PD-related neurodegeneration. However, whether inflammation may cause oxidative damage in MPTP and PD is unknown. Here we show that NADPH-oxidase, the main reactive oxygen species (ROS)-producing enzyme during inflammation, is up-regulated in SNpc of human PD and MPTP mice. These changes coincide with the local production of ROS, microglial activation, and DA neuronal loss seen after MPTP injections. Mutant mice defective in NADPH-oxidase exhibit less SNpc DA neuronal loss and protein oxidation than their WT littermates after MPTP injections. We show that extracellular ROS are a main determinant in inflammation-mediated DA neurotoxicity in the MPTP model of PD. This study supports a critical role for NADPH-oxidase in the pathogenesis of PD and suggests that targeting this enzyme or enhancing extracellular antioxidants may provide novel therapies for PD.

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Figures

Figure 1

Figure 1

(A_–_D) RT-PCR shows ventral midbrain gp91phox, p67phox, and Mac-1 mRNA levels in saline-injected (S) and MPTP-injected mice from 0 to 14 days after injections. SNpc gp91phox mRNA labeling is negligible in saline-injected mice (E), whereas it is copious in MPTP-injected mice at 2 days (F). *, P < 0.05; **, P < 0.001, more than saline-treated mice (n = 4–6 per time point). (Scale, 2.5 mm.)

Figure 2

Figure 2

Western blot shows the time-dependent induction of gp91phox in mouse ventral midbrain after MPTP injections. +, Mouse macrophage lysate; s, saline. In saline-injected mice, gp91phox immunoreactivity (C and D, brown) is mild and localized in resting microglia, which are not abundant in the SNpc, as shown (E and F) by Mac-1 labeling (brown, arrow) and are intermingled with TH-positive neurons (gray-blue). Two days after MPTP injections, numerous gp91phox-positive cells are seen in the SNpc (G and H). These cells resemble activated microglial cells (H vs. J, arrow). At this point there are many fewer TH-positive neurons (I and J, arrowhead). Confocal microscopy shows that all gp91phox-positive cells are Mac-1-positive, thus confirming their microglial origin (K and L). Conversely, no gp91phox-positive cells are glial fibrillary acidic protein-positive cells, thus excluding their astrocyctic origin (N–P). *, P < 0.05, more than saline-treated mice (n = 6 per time point). [Scale bar, 2.5 mm (C, E, G, and I); 0.25 mm (D, F, H, and J); and 0.2 mm (K_–_P).]

Figure 3

Figure 3

(A) Representative Western blots illustrating the increase in ventral midbrain gp91phox protein content in two PD and two controls. (B) Bar graph showing mean Western blot gp91phox/β-actin ratios ± SEM for six PD and three control ventral midbrain samples. (C and D) Representative gp91phox immunostaining that shows positive cells in PD samples (arrowhead, gray-blue, membrane labeling) colocalizing with the microglial marker CD68 (arrow, red, cytosol labeling), but not with neuromelanin (brown pigment). *, P < 0.05, higher than controls. (Scale bar, 0.5 mm.)

Figure 4

Figure 4

Ethidium fluorescence (A) and Mac-1 immunostaining (B) are minimal in the saline-treated mice. By 2 days after MPTP injections, SNpc ethidium fluorescence is increased in WT mice (C) and is absent in gp91phox-deficient mice (E) and minocycline-treated WT mice (G). Microglial activation is prevented by minocycline (H) but is normal in gp91phox-deficient mice (E). MPTP stimulates NADPH-oxidase activation, as evidenced by p67phox translocation from the cytosol to the plasma membrane in WT mice (wt), but not in gp91phox-deficient mice (ko) (I and J); the membrane protein calnexin is used to normalize the data. Data are means ± SEM for four to six samples per group. *, P < 0.05, higher than controls; #, P < 0.05, less than MPTP-injected WT mice, but not different from both saline-injected groups.

Figure 5

Figure 5

Stereological counts of TH-positive neurons in the SNpc (A) and optical density of striatal DA fibers (B) are higher in gp91phox-deficient mice (ko) compared with their WT littermates (wt) 7 days after MPTP injections (n = 4–8 samples per group). *, P < 0.05, less than saline-injected mice; #, P < 0.05, higher than MPTP-injected WT mice.

Figure 6

Figure 6

(A) Ventral midbrain carbonyl content, used as a marker of protein oxidative damage, is increased at 2 days after MPTP injections in WT mice (wt), but not in gp91phox-deficient mice (ko). Infusion of SOD1 into the left striatum attenuates the striatal (B) and the SNpc lesion on the infused side, but not on the contralateral, noninfused side (C) after a systemic injection of MPTP. *, P < 0.05, higher than controls; #, P < 0.05, less than MPTP-injected WT mice, but not different from the two saline-injected groups.

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References

    1. Fahn S, Przedborski S. In: Merritt's Neurology. Rowland L P, editor. New York: Lippincott; 2000. pp. 679–693.
    1. Przedborski S, Kostic V, Giladi N, Eidelberg D. In: Dopamine Receptors and Transporters. Sidhu A, Laruelle M, Vernier P, editors. New York: Dekker; 2003. pp. 363–402.
    1. Chen H, Zhang S M, Hernan M A, Schwarzschild M A, Willett W C, Colditz G A. Movement Disorders. 2002;17,(Suppl. 5):S143.
    1. Gao H M, Jiang J, Wilson B, Zhang W, Hong J S, Liu B. J Neurochem. 2002;81:1285–1297. - PubMed
    1. Wu D C, Jackson-Lewis V, Vila M, Tieu K, Teismann P, Vadseth C, Choi D K, Ischiropoulos H, Przedborski S. J Neurosci. 2002;22:1763–1771. - PMC - PubMed

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