Pharmacological inhibition of neuronal NADPH oxidase protects against 1-methyl-4-phenylpyridinium (MPP+)-induced oxidative stress and apoptosis in mesencephalic dopaminergic neuronal cells.
Oxidative stress is widely recognized as a key mediator of degenerative processes in Parkinson's disease (PD). Recently, we demonstrated that the dopaminergic toxin MPP+ initiates oxidative stress to cause caspase-3-dependent apoptotic cell death in mesencephalic dopaminergic neuronal (N27) cells. In this study, we determined the source of reactive oxygen species (ROS) produced during MPP+-induced apoptotic cell death. In addition to mitochondria, plasma membrane NADPH oxidase is considered a major producer of ROS inside the cell. Here, we show that N27 neuronal cells express key NADPH oxidase subunits gp91phox and p67phox. We used structurally diverse NADPH oxidase inhibitors, aminoethyl-benzenesulfonylfluoride (AEBSF, 100-1000microM), apocynin (100-1000microM), and diphenylene iodonium (DPI, 3-30microM), to inhibit intrinsic NADPH oxidase activity in N27 cells. Flow cytometric analysis using the ROS-sensitive dye hydroethidine revealed that AEBSF blocked 300microM MPP+-induced ROS production for over 45min in N27 cells, in a dose-dependent manner. Further treatment with DPI, apocynin, and SOD also blocked MPP+-induced ROS production. In Sytox cell death assays, co-treatment with AEBSF, apocynin, or DPI for 24h significantly suppressed MPP+-induced cytotoxic cell death. Similarly, co-treatment with these inhibitors also significantly attenuated MPP+-induced increases in caspase-3 enzymatic activity. Furthermore, quantitative DNA fragmentation ELISA assays revealed that AEBSF, DPI, and apocynin rescue N27 cells from MPP+-induced apoptotic cell death. Together, these results indicate for the first time that intracellular ROS generated by NAPDH oxidase are present within the mesencephalic neuronal cells, and are a key determinant of MPP+-mediated dopaminergic degeneration in in vitro models of dopaminergic degeneration. This study supports a critical role of NADPH oxidase in the oxidative damage in PD; targeting this enzyme may lead to novel therapies for PD.
Anantharam
Vellareddy
V
Parkinson's Disorder Research Laboratory, Iowa Center for Advanced Neurotoxicology, Department of Biomedical Sciences, Iowa State University, Ames, IA 50011-1250, USA.
Kaul
Siddharth
S
Song
Chunjuan
C
Kanthasamy
Arthi
A
Kanthasamy
Anumantha G
AG
D015655:1-Methyl-4-phenylpyridinium / Q000037:antagonists & inhibitors* / Q000633:toxicity*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D004298:Dopamine / Q000502:physiology*; D015259:Dopamine Agents / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D020267:MPTP Poisoning / Q000378:metabolism* / Q000517:prevention & control*; D008562:Membrane Glycoproteins / Q000235:genetics; D008636:Mesencephalon / Q000166:cytology / Q000187:drug effects*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000235:genetics; D009474:Neurons / Q000187:drug effects / Q000201:enzymology*; D009930:Organic Chemicals; D018384:Oxidative Stress / Q000187:drug effects*; D010750:Phosphoproteins / Q000235:genetics; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism
D015259:Dopamine Agents; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D009930:Organic Chemicals; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; C402795:SYTOX Green; C079789:neutrophil cytosol factor 67K; C500593:Cybb protein, rat; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D020169:Caspases; D015655:1-Methyl-4-phenylpyridinium; D004298:Dopamine