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, 138 (Pt 5), 1247-62

Post-treatment With an Ultra-Low Dose of NADPH Oxidase Inhibitor Diphenyleneiodonium Attenuates Disease Progression in Multiple Parkinson's Disease Models

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Post-treatment With an Ultra-Low Dose of NADPH Oxidase Inhibitor Diphenyleneiodonium Attenuates Disease Progression in Multiple Parkinson's Disease Models

Qingshan Wang et al. Brain.

Abstract

Nicotinamide adenine dinucleotide phosphate oxidase, a key superoxide-producing enzyme, plays a critical role in microglia-mediated chronic neuroinflammation and subsequent progressive dopaminergic neurodegeneration in Parkinson's disease. Although nicotinamide adenine dinucleotide phosphate oxidase-targeting anti-inflammatory therapy for Parkinson's disease has been proposed, its application in translational research remains limited. The aim of this study was to obtain preclinical evidence supporting this therapeutic strategy by testing the efficacy of an ultra-low dose of the nicotinamide adenine dinucleotide phosphate oxidase inhibitor diphenyleneiodonium in both endotoxin (lipopolysaccharide)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice using post-treatment regimens. Our data revealed that post-treatment with diphenyleneiodonium significantly attenuated progressive dopaminergic degeneration and improved rotarod activity. Remarkably, post-treatment with diphenyleneiodonium 10 months after lipopolysaccharide injection when mice had 30% loss of nigral dopaminergic neurons, showed high efficacy in protecting the remaining neuronal population and restoring motor function. Diphenyleneiodonium-elicited neuroprotection was associated with the inhibition of microglial activation, a reduction in the expression of proinflammatory factors and an attenuation of α-synuclein aggregation. A pathophysiological evaluation of diphenyleneiodonium-treated mice, including assessment of body weight, organs health, and neuronal counts, revealed no overt signs of toxicity. In summary, infusion of ultra-low dose diphenyleneiodonium potently reduced microglia-mediated chronic neuroinflammation by selectively inhibiting nicotinamide adenine dinucleotide phosphate oxidase and halted the progression of neurodegeneration in mouse models of Parkinson's disease. The robust neuroprotective effects and lack of apparent toxic side effects suggest that diphenyleneiodonium at ultra-low dose may be a promising candidate for future clinical trials in Parkinson's disease patients.

Keywords: NADPH oxidase; Parkinson’s disease; microglia; neuroinflammation; superoxide.

Figures

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Oxidative stress has been implicated in Parkinson’s disease progression. Wang et al. show that treatment with ultra-low doses of the NADPH oxidase inhibitor diphenyleneiodonium reverses parkinsonian defects and halts progression in three mouse models of the disease, even when treatment is started when over 30% of dopaminergic neurons are lost.
Figure 1
Figure 1
Post-treatment with an ultra-low dose of DPI attenuates dopaminergic neurodegeneration and motor deficits in LPS-treated mice. (A) Experimental designs. C57BL/6J mice received a single injection of LPS [15 × 106 EU/kg, intraperitoneally (i.p.)]. Three (pre-motor stage) or 10 (motor stage) months after LPS injection, the mice were infused with either vehicle or DPI (10 ng/kg/day; subcutaneously) via osmotic mini-pump for 2 weeks. Measurements of neuron loss and motor deficits were performed 7 months after DPI infusion. (B) Seven months after DPI treatment, dopaminergic neurons in the substantia nigra pars compacta were immunostained with anti-tyrosine hydroxylase antibody and representative images are shown. (C) The number of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra pars compacta was counted stereologically. (D) The effects of ultra-low-dose DPI on LPS-induced motor deficits were measured using the rotarod test. Data are expressed as the mean ± SEM and were analysed by two-way ANOVA followed by Tukey’s post hoc testing. *P < 0.05, **P < 0.01; #P < 0.05, ##P < 0.01; n = 6–11; Scale bar = 200 µm. Con = control; TH = tyrosine hydroxylase; THir = tyrosine hydroxylase-immunoreactive.
Figure 2
Figure 2
Post-treatment with an ultra-low dose of DPI protects dopaminergic neurons against LPS-induced damage in transgenic mice over-expressing human A53T mutant α-synuclein. (A) Experimental designs. Seven-month-old transgenic mice over-expressing human A53T mutant α-synuclein received a single injection of LPS [6 × 106 EU/kg, intraperitoneally (i.p.)]. One month after LPS injection, the mice were infused with either vehicle or DPI (10 ng/kg/day; subcutaneously) via osmotic mini-pump for 2 weeks. (B) Three months after DPI post-treatment, nigral dopaminergic neurons and striatal axon fibres were immunostained with anti-tyrosine hydroxylase antibody, and representative images are shown. (C) The number of tyrosine hydroxylase-immunoreactive cells was counted stereologically, and the results are expressed as the mean ± SEM. (D) The density of tyrosine hydroxylase immunostaining in the striatum was quantified using densitometric analysis. Data were analysed by one-way ANOVA followed by Tukey’s post hoc testing. **P < 0.01; Scale bar = 200 µm; n = 5–6. TH = tyrosine hydroxylase.
Figure 3
Figure 3
Post-treatment with an ultra-low dose of DPI attenuates nigral α-synuclein aggregation in LPS-treated transgenic mice over-expressing human A53T mutant α-synuclein. (A) One month after LPS injection, transgenic mice over-expressing human A53T mutant α-synuclein were infused with either vehicle or DPI (10 ng/kg/day; subcutaneously) via osmotic mini-pump for 2 weeks. Three months after DPI post-treatment, human α-synuclein was immunostained in the substantia nigra with SYN211 (specific for human α-synuclein) antibody, and representative images are shown. (B) Magnifications of dopaminergic neuron (tyrosine hydroxylase-immunoreactive) and α-synuclein double staining are indicated in the different groups. (C) The SYN211 density in the substantia nigra was quantified. The results are expressed as a percentage of the vehicle controls (mean ± SEM) and were analysed by one-way ANOVA followed by Tukey’s post hoc testing. **P < 0.01; Scale bar = 200 µm in (A) and 50 µm in (B); n = 5–6. α-Syn = α-synuclein.
Figure 4
Figure 4
Post-treatment with an ultra-low dose of DPI attenuates chronic microglial activation. (A) Three (pre-motor) or 10 (motor) months after LPS injection, mice (C57BL/6J) were infused with either vehicle or DPI (10 ng/kg/day; subcutaneously) via osmotic mini-pump for 2 weeks. Two microglial markers, ITGAM (CD11b) or AIF1 (Iba-1), were immunostained in the substantia nigra region 7 months after DPI treatment. Representative pictures of staining are shown. Activated microglia characterized by an enlarged cell body size and high staining density. (BD) The activation of microglia was quantified by measuring the density of ITGAM (CD11b; B) and AIF1 (Iba-1) (C) and the cell body size (D). The gene expressions of tumor necrosis factor alpha (TNFα; E), interleukin-1 beta (Il-1β; F) and major histocompatibility complex II (G) in brain were determined in the rostral half of the brains using RT-PCR. Data are expressed as a percentage of time-matched vehicle controls (mean ± SEM) and were analysed by two-way ANOVA followed by Tukey’s post hoc testing. *P < 0.05, **P < 0.01; n = 5–6; Scale bar = 50 µm. MHCII = major histocompatibility complex II.
Figure 5
Figure 5
Post-treatment with an ultra-low dose of DPI inhibits the LPS-induced increase in dihydroxyethidium oxidation but not the activity of mitochondrial complex I. (A) Experimental designs. Ten months after LPS injection, mice (C57BL/6J) were infused with DPI (10 ng/kg/day). After 2 weeks of DPI infusion, the brains were perfused, and the production of superoxide was determined by measuring the oxidation products of dihydroethidium by fluorescence microscopy. (B) Representative images of dihydroethidium staining (red) are shown. The position of the substantia nigra was identified by tyrosine hydroxylase staining (green). (C) Magnification of dopaminergic neurons (tyrosine hydroxylase-immunoreactive) and dihydroethidium double staining are indicated in the different groups. (D) Quantified data showing the fluorescence density of the dihydroethidium oxidation in the substantia nigra of different treatment groups. Fluorescence density of NADPH oxidase-deficient (gp91phox−/−) mice served as a control background. (E) The effects of ultra-low-dose DPI on the activities of complex I in the brain were determined using commercial assay kits. Data are expressed as a percentage of time-matched vehicle controls (mean ± SEM) and were analysed by Student’s t-test (E) or one-way ANOVA followed by Tukey’s post hoc testing (D). **P < 0.01; n = 5; Scale bar = 100 µm. DHE = dihydroethidium; TH = tyrosine hydroxylase; THir = tyrosine hydroxylase-immunoreactive.
Figure 6
Figure 6
Post-treatment with an ultra-low dose of DPI attenuates MPTP-induced dopaminergic neuron damage and motor deficits. (A) Experimental designs. Repeated MPTP regimens (15 mg/kg, subcutaneously for 6 consecutive days) were administered to C57BL/6J mice. After 3 days of MPTP injection, the mice were infused with either vehicle or DPI (10 ng/kg/day; subcutaneously) via osmotic mini-pump for 2 weeks. (B) Twenty-seven days after the first injection of MPTP, dopaminergic neurons in the substantia nigra pars compacta were immunostained with anti-tyrosine hydroxylase antibody, and the numbers of tyrosine hydroxylase-immunoreactive cells were counted. (C) The turnover rate of dopamine in the striatum was calculated by the ratio of dopamine metabolite (DOPAC) and dopamine. (D) The protective effects of DPI against MPTP-induced motor deficits were measured by the rotarod test. Data are expressed as the mean ± SEM and were analyzed by one-way ANOVA followed by Tukey’s post hoc testing. *P < 0.05, **P < 0.01; n = 8–10. TH = tyrosine hydroxylase; THir = tyrosine hydroxylase-immunoreactive.
Figure 7
Figure 7
An ultra-low dose of DPI displays no toxicity. Mice were treated with either vehicle or DPI (10 ng/kg/day; subcutaneously) using an Alzet mini-pump for 2 weeks. The effects of ultra-low-dose DPI on rotarod (A), and body (B), liver (C), spleen (D) and kidney (E) weights were assessed. (F) Organs including liver, spleen, kidney, testis, heart, lung and brain were dissected and stained with haematoxylin and eosin. Representative images are shown. Data were analysed using a Student’s t-test. n = 3; Scale bar = 100 µm.
Figure 8
Figure 8
Proposed model showing how ultra-low-dose DPI attenuates progressive dopaminergic neurodegeneration. NADPH oxidase is a key mediator for initiating and maintaining the self-propagating vicious cycle formed between damaged neurons and dysregulated microglia. The self-propelling vicious cycle is critical in driving the progressive dopaminergic degeneration in Parkinson’s disease. Ultra-low dose DPI is capable of inhibiting the activation of NADPH oxidase and subsequent production of superoxide and other neurotoxic factors to mitigate chronic neuroinflammation. Once the self-propelling vicious cycle is interrupted by inhibiting NADPH oxidase on microglia, neurons or both, the progression of dopaminergic neuron degeneration can be halted. These results provide a novel and promising avenue for developing drug therapy for Parkinson’s disease and other neurodegenerative diseases.

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