title: Subpicomolar diphenyleneiodonium inhibits microglial NADPH oxidase with high specificity and shows great potential as a therapeutic agent for neurodegenerative diseases.
abstract: Activation of microglial NADPH oxidase (NOX2) plays a critical role in mediating neuroinflammation, which is closely linked with the pathogenesis of a variety of neurodegenerative diseases, including Parkinson's disease (PD). The inhibition of NOX2-generated superoxide has become an effective strategy for developing disease-modifying therapies for PD. However, the lack of specific and potent NOX2 inhibitors has hampered the progress of this approach. Diphenyleneiodonium (DPI) is a widely used, long-acting NOX2 inhibitor. However, due to its non-specificity for NOX2 and high cytotoxicity at standard doses (µM), DPI has been precluded from human studies. In this study, using ultra-low doses of DPI, we aimed to: (1) investigate whether these problems could be circumvented and (2) determine whether ultra-low doses of DPI were able to preserve its utility as a potent NOX2 inhibitor. We found that DPI at subpicomolar concentrations (10(-14) and 10(-13) M) displays no toxicity in primary midbrain neuron-glia cultures. More importantly, we observed that subpicomolar DPI inhibited phorbol myristate acetate (PMA)-induced activation of NOX2. The same concentrations of DPI did not inhibit the activities of a series of flavoprotein-containing enzymes. Furthermore, potent neuroprotective efficacy was demonstrated in a post-treatment study. When subpicomolar DPI was added to neuron-glia cultures pretreated with lipopolysaccharide, 1-methyl-4-phenylpyridinium or rotenone, it potently protected the dopaminergic neurons. In summary, DPI's unique combination of high specificity toward NOX2, low cytotoxicity and potent neuroprotective efficacy in post-treatment regimens suggests that subpicomolar DPI may be an ideal candidate for further animal studies and potential clinical trials.
authors.affiliation: Neuropharmacology Section, Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina.
authors.forename: Qingshan
authors.initials: Q
authors.lastname: Wang
authors.forename: Chun-Hsien
authors.initials: CH
authors.lastname: Chu
authors.forename: Esteban
authors.initials: E
authors.lastname: Oyarzabal
authors.forename: Lulu
authors.initials: L
authors.lastname: Jiang
authors.forename: Shih-Heng
authors.initials: SH
authors.lastname: Chen
authors.forename: Belinda
authors.initials: B
authors.lastname: Wilson
authors.forename: Li
authors.initials: L
authors.lastname: Qian
authors.forename: Jau-Shyong
authors.initials: JS
authors.lastname: Hong
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002452:Cell Count; D002478:Cells, Cultured; D004298:Dopamine / Q000378:metabolism; D042967:Electron Transport Complex I / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D008636:Mesencephalon / Q000166:cytology; D008840:Microfilament Proteins / Q000378:metabolism; D017628:Microglia / Q000187:drug effects*; D019255:NADPH Oxidases / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology*; D013481:Superoxides / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D014969:Xanthine Oxidase / Q000378:metabolism
chemical_list: C100745:Aif1 protein, rat; D002135:Calcium-Binding Proteins; D004791:Enzyme Inhibitors; D005904:Glial Fibrillary Acidic Protein; D008840:Microfilament Proteins; D009573:Nitrites; D009861:Onium Compounds; D013481:Superoxides; C007517:diphenyleneiodonium; D052247:Nitric Oxide Synthase Type II; D014446:Tyrosine 3-Monooxygenase; D014969:Xanthine Oxidase; D009251:NADPH-Ferrihemoprotein Reductase; D019255:NADPH Oxidases; D013880:Thioredoxin-Disulfide Reductase; D042967:Electron Transport Complex I; D004298:Dopamine
keywords: NADPH oxidase; Parkinson's disease; microglia; neuroinflammation; oxidative stress