NADPH Oxidase Mediates β-Amyloid Peptide-Induced Neuronal Death in Mouse Cortical Cultures
10.4068/cmj.2017.53.3.196
- Author:
Kee Oh CHAY
1
;
Kyoung Young NAM KOONG
;
Shinae HWANG
;
Jong Keun KIM
;
Choon Sang BAE
Author Information
1. Department of Biochemistry, Chonnam National University Medical School, Gwangju, Korea.
- Publication Type:Original Article
- Keywords:
Amyloid beta-Peptides;
Alzheimer Disease;
NADPH Oxidase;
Reactive Oxygen Species
- MeSH:
Alzheimer Disease;
Amyloid beta-Peptides;
Animals;
Antioxidants;
Ascorbic Acid;
Astrocytes;
Baths;
Blotting, Western;
Cell Death;
Cycloheximide;
Fluorescence;
Humans;
L-Lactate Dehydrogenase;
Mice;
Microglia;
NADP;
NADPH Oxidase;
Neurons;
Plaque, Amyloid;
Reactive Oxygen Species
- From:Chonnam Medical Journal
2017;53(3):196-202
- CountryRepublic of Korea
- Language:English
-
Abstract:
β-Amyloid peptide (Aβ) is the main component of senile plaques in patients with Alzheimer's disease, and is known to be a main pathogenic factor of the disease. Recent evidence indicates that activation of NADPH oxidase (NOX) in microglia or astrocytes may be a source of Aβ-induced reactive oxygen species (ROS). We investigated the role of neuronal NOX in Aβ-induced neuronal death in mouse mixed cortical cultures. Cell death was assessed by measuring lactate dehydrogenase efflux to bathing media 24 or 48 hr after exposure to Aβ₂₅₋₃₅, a fragment of Aβ with an equivalent neurotoxic effect. Aβ₂₅₋₃₅ induced neuronal death in concentration- and time- dependent manners with apoptotic features. Neuronal death was significantly attenuated, not only by anti-apoptotic drugs, such as z-VAD-fmk and cycloheximide, but also by antioxidants, such as trolox, ascorbic acid, and epigallocatethin gallate. We also demonstrated that treatment with 20 µM Aβ₂₅₋₃₅ increased fluorescent signals in mixed cortical cultures, but produced only weak signals in pure astrocyte cultures in the presence of 2',7'-dichlorofluorescin diacetate (DCF-DA), an indicator for intracellular ROS. Increased DCF-DA fluorescence was markedly inhibited, not only by trolox, but also by selective NOX inhibitors, such as apocynin and AEBSF. Western blot analyses revealed that Aβ₂₅₋₃₅ increased the expression of gp91phox, a main subunit of NOX in cells. The above antioxidants, apocynin, and AEBSF significantly attenuated neuronal death induced by Aβ₂₅₋₃₅. Furthermore, the gp91phox-specific siRNA-based knockdown of NOX significantly inhibited neuronal death. These results suggest that activation of neuronal NOX is involved in Aβ25-35-induced neuronal death.
