Chonnam Med J.  2017 Sep;53(3):196-202. 10.4068/cmj.2017.53.3.196.

NADPH Oxidase Mediates β-Amyloid Peptide-Induced Neuronal Death in Mouse Cortical Cultures

• Chay KO
• Nam Koong
• Hwang S
• Kim JK
• Bae CS

• KMID: 2390433
• DOI: http://doi.org/10.4068/cmj.2017.53.3.196

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.

Keyword

Amyloid beta-Peptides; Alzheimer Disease; NADPH Oxidase; Reactive Oxygen Species

MeSH Terms

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
Amyloid beta-Peptides
Antioxidants
Ascorbic Acid
Cycloheximide
L-Lactate Dehydrogenase
NADP
NADPH Oxidase
Reactive Oxygen Species

Figure

• 

  FIG. 1 β-Amyloid peptide (Aβ)25-35-induced neuronal death in mouse cortical cultures and the protective effects of antioxidants. (A) Cell death measured by assay for leaked lactate dehydrogenase (LDH) activity in the media, showing the LDH activity of the NMDA-treated cell group (% LDH release). Each column and bar are the mean±SEM from 8-20 wells. (B) Fluorescent photomicrographs from typical representative fields (200×field) of cells were taken after a 24-hour exposure to sham wash (sham) or 20 µM Aβ25-35 (Aβ). Arrows indicate fragmented and condensed chromatin stained with SYTOX green. (C) Inhibitory effect of 1 µg/mL cycloheximide (+ CHX), or 100 µM z-VAD-fmk (+ZVAD), on the 20 µM Aβ25-35-induced neuronal death after 24 and 48 hr of exposure. Each column and bar is the mean±SEM from 12-16 wells. (D) Effect of co-treatment with trolox (100 µM), ascorbic acid (100, 2000 µM) or EGCG (10, 30 µM) on the 20 µM Aβ25-35-induced neuronal death after 24 and 48 hr of exposure. Each column and bar are the mean±SEM from 8-12 wells. *Significantly different from corresponding Aβ-treated control group (p<0.05).

• 

  FIG. 2 Time-course of reactive oxygen species (ROS) generation by 20 µM Aβ25-35 and the effects of treatment with apocynin (1 mM), AEBSF (50 µM), and trolox (100 µM) on Aβ25-35-induced ROS generation in mouse neuron-glia mixed cortical (mixed) and astrocyte (Astrocyte) cultures. ROS generation was measured as the relative strength of fluorescence (Ex/Em=485 nm/538 nm) from the Aβ25-35-exposed cells in the presence of 2',7'-dichlorofluorescin diacetate dye. *Significantly different from negative or Aβ control groups (p<0.05).

• 

  FIG. 3 Induction of gp91phox in mixed cortical cultures exposed to 20 µM Aβ25-35. Western blots for gp91phox in sham-washed control (basal) and sister cultures 1, 2 and 4 hr after exposure to 20 µM Aβ25-35 (Aβ). The panels are representative of three independent experiments.

• 

  FIG. 4 Protective effects of co-treatment with apocynin (500 µM or 1 mM) or 50 µM AEBSF on 20 µM Aβ25-35-induced neuronal death after 24 and 48 hr of exposure. Cell death was measured as described in Fig. 1. Each column and bar are the mean±SEM from 8-16 wells. *Significantly different from corresponding Aβ-treated control group (p<0.05).

• 

  FIG. 5 Effects of knockdown of gp91phox with siRNA on 20 µM and 40 µM Aβ25-35-induced neuronal death after 24 hr of exposure. Upper representative western blots for gp91phox in sham-washed control (basal) and sister cultures 24 hr after treatment with mock siRNA (negative) and siRNA. Negative represents the scrambled mock siRNA treated group. Cell death was measured as described in FIG. 1. Each column and bar are the mean±SEM from 8-16 wells. *Significantly different from negative or Aβ control groups (p<0.05).

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