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Cited by 683Alzheimer's disease research and treatmentsMitochondrial Function and PathologyRedox biology and oxidative stress

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NOX4 promotes ferroptosis of astrocytes by oxidative stress-induced lipid peroxidation via the impairment of mitochondrial metabolism in Alzheimer’s diseases

Open access
Park, Min Woo, Cha, Hyeon Woo, Kim, Junhyung, Kim, Jung Han, Yang, Haesung, Yoon, Sunmi, Boonpraman, Napissara, Yi, Sun Shin, Yoo, Ik Dong, Moon, Jong-Seok·2021·Redox Biology
DOI 10.1016/j.redox.2021.101947PMC PMC8027773
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Abstract

Oxidative stress has been implicated in the pathogenesis of Alzheimer's disease (AD). Mitochondrial dysfunction is linked to oxidative stress and reactive oxygen species (ROS) in neurotoxicity during AD. Impaired mitochondrial metabolism has been associated with mitochondrial dysfunction in brain damage of AD. While the role of NADPH oxidase 4 (NOX4), a major source of ROS, has been identified in brain damage, the mechanism by which NOX4 regulates ferroptosis of astrocytes in AD remains unclear. Here, we show that the protein levels of NOX4 were significantly elevated in impaired astrocytes of cerebral cortex from patients with AD and APP/PS1 double-transgenic mouse model of AD. The levels of 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), a marker of oxidative stress-induced lipid peroxidation, were significantly also elevated in impaired astrocytes of patients with AD and mouse AD. We demonstrate that the over-expression of NOX4 significantly increases the impairment of mitochondrial metabolism by inhibition of mitochondrial respiration and ATP production via the reduction of five protein complexes in the mitochondrial ETC in human astrocytes. Moreover, the elevation of NOX4 induces oxidative stress by mitochondrial ROS (mtROS) production, mitochondrial fragmentation, and inhibition of cellular antioxidant process in human astrocytes. Furthermore, the elevation of NOX4 increased ferroptosis-dependent cytotoxicity by the activation of oxidative stress-induced lipid peroxidation in human astrocytes. These results suggest that NOX4 promotes ferroptosis of astrocytes by oxidative stress-induced lipid peroxidation via the impairment of mitochondrial metabolism in AD.

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TL;DR

NOX4 protein is elevated in impaired astrocytes in AD patient cortex and APP/PS1 mouse cortex, where lipid peroxidation markers 4-HNE and MDA are also increased. In human astrocytes, NOX4 overexpression impairs mitochondrial metabolism, induces oxidative stress and antioxidant suppression, and promotes ferroptosis-like lipid peroxidation and cytotoxicity.

Key findings

  • NOX4 levels were significantly elevated in GFAP-positive impaired astrocytes in AD cortex versus non-AD cortex (n = 3 per group, n = 10 images per subject; p < 0.01) and in APP/PS1 mouse cortex versus WT (n = 5 per group, n = 10 images per subject; p < 0.01).
  • Oxidative stress-induced lipid peroxidation markers 4-HNE and MDA were significantly elevated in impaired astrocytes of AD patients and APP/PS1 mice, and 4-HNE co-localized with NOX4-positive astrocytes (p < 0.01).
  • In human astrocytes, NOX4 overexpression significantly suppressed basal and oligomycin/FCCP-responsive oxygen consumption rate, reduced mitochondrial ATP production, and reduced protein levels of all five mitochondrial ETC complexes: Complex I NDUFB8, Complex II SDHB, Complex III UQCRC2, Complex IV MTCO1, and Complex V ATP5F1A (p < 0.05 or p < 0.01).
  • NOX4 overexpression increased mtROS and mitochondrial fragmentation, reduced GSH and GSH/GSSG, increased GSSG, inhibited NRF2 nuclear translocation and HO-1/GCL, and increased 4-HNE, MDA, iron accumulation, morphological cell death, and LDH cytotoxicity (p < 0.05 or p < 0.01).

Methods

  • Postmortem human brain tissues: 12 paraffin-embedded tissues from 3 AD donors (frontal, occipital, temporal, parietal cortex) and 4 normal adult brain tissues from 4 donors; immunofluorescence analyzed NOX4, 4-HNE, MDA, GFAP, and Tomm20. APP/PS1 double-transgenic mice were compared with WT mice.
  • Human astrocytes were transduced with pCMV6-AC-GFP-NOX4 or vector. Assays included Seahorse XF96e OCR and real-time ATP rate, immunoblot for mitochondrial ETC complexes, MitoSOX flow for mtROS, GSH/GSSG and GSSG measurements, NRF2 nuclear/cytosolic fractionation, HO-1 and GCL levels/activity, iron colorimetric assay, LDH cytotoxicity, and 3D imaging.
  • Statistics used Shapiro-Wilk and Levene’s tests, two-tailed Student’s t-test for two groups, and ANOVA with Dunnett’s post hoc for multiple groups; significance was p < 0.05, p < 0.01, or p < 0.001.

Limitations

  • Stated: it remains unclear whether the impaired astrocytes with high NOX4 have reactive astrocyte function in AD, and further study is needed on the role of NOX4 in reactive astrocytes. The authors also state that further study is needed to identify secreted molecules from NOX4-induced ferroptosis as potential diagnostic markers.
  • Evident from the text: the human sample is small (3 AD donors and 4 normal donors), and the mechanistic causality in human astrocytes relies mainly on NOX4 overexpression; the text does not report NOX4 knockdown, NOX4 inhibition, or ferroptosis-blockade rescue experiments.
  • Evident from the text: APP/PS1 mouse data are descriptive immunofluorescence comparisons, without in vivo NOX4 manipulation reported.

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Registry

Crossref · 10.1016/j.redox.2021.101947

Cites

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Figures

Fig. 1 The levels of NOX4 are elevated in impaired astrocytes of the cortex region from patients with Alzheimer's diseases. (A) Representative immunofluorescence images of NOX4 protein expression in cerebral cortex region from patients with AD (AD #1, AD #2, AD #3) or non-AD (normal) showing NOX4 (green) in astrocytes expressing astrocytes marker GFAP (red) around molecular layer (ML) (n = 3 per group, n = 10 images per individual subject). DAPI-stained nuclei are shown in blue. OS, Outer surface; ML, Molecular layer; EGL, External granular layer. Scale bars, 20 μm. White arrows indicate NOX4 and GFAP positive cells. Symbols, which are expressed by white dotted line, indicate the distinct area among OS, ML, and EGL. (B) Quantification of intensity for NOX4 positive staining in astrocytes from immunofluorescence images in the cerebral cortex region from patients with AD (AD #1, AD #2, AD #3) or non-AD (normal) (n = 3 per group, n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (C) Quantification of NOX4 positive astrocytes from immunofluorescence images in the cerebral cortex region from patients with AD (AD #1, AD #2, AD #3) or non-AD (normal) (n = 3 per group, n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 1 The levels of NOX4 are elevated in impaired astrocytes of the cortex region from patients with Alzheimer's diseases. (A) Representative immunofluorescence images of NOX4 protein expression in cerebral cortex region from patients with AD (AD #1, AD #2, AD #3) or non-AD (normal) showing NOX4 (green) in astrocytes expressing astrocytes marker GFAP (red) around molecular layer (ML) (n = 3 per group, n = 10 images per individual subject). DAPI-stained nuclei are shown in blue. OS, Outer surface; ML, Molecular layer; EGL, External granular layer. Scale bars, 20 μm. White arrows indicate NOX4 and GFAP positive cells. Symbols, which are expressed by white dotted line, indicate the distinct area among OS, ML, and EGL. (B) Quantification of intensity for NOX4 positive staining in astrocytes from immunofluorescence images in the cerebral cortex region from patients with AD (AD #1, AD #2, AD #3) or non-AD (normal) (n = 3 per group, n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (C) Quantification of NOX4 positive astrocytes from immunofluorescence images in the cerebral cortex region from patients with AD (AD #1, AD #2, AD #3) or non-AD (normal) (n = 3 per group, n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)CC BY-NC-ND · PubMed Central
Fig. 2 The levels of oxidative stress-induced lipid peroxidation are elevated in impaired astrocytes of the cortex region from patients with Alzheimer's diseases. (A) Representative immunofluorescence images of 4-HNE protein expression in cerebral cortex region from patients with AD (AD #1, AD #2, AD #3) or non-AD (normal) showing 4-HNE (green) in astrocytes expressing astrocytes marker GFAP (red) around molecular layer (ML) (n = 3 per group, n = 10 images per individual subject). DAPI-stained nuclei are shown in blue. OS, Outer surface; ML, Molecular layer; EGL, External granular layer. Scale bars, 20 μm. White arrows indicate 4-HNE and GFAP positive cells. Symbols, which are expressed by white dotted line, indicate the distinct area among OS, ML, and EGL. (B) Quantification of intensity of 4-HNE positive staining in astrocytes from immunofluorescence images in the cerebral cortex region from patients with AD (AD #1, AD #2, AD #3) or non-AD (normal) (n = 3 per group, n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (C) Quantification of 4-HNE positive astrocytes from immunofluorescence images in the cerebral cortex region from patients with AD (AD #1, AD #2, AD #3) or non-AD (normal) (n = 3 per group, n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (D) Representative immunofluorescence images of 4-HNE protein expression in NOX4-positive astrocytes of patients with AD (AD) or non-AD (normal) showing 4-HNE (purple) in NOX4 (green)-positive astrocytes expressing astrocytes marker GFAP (red) around molecular layer (ML) (n = 10 images per individual subject). DAPI-stained nuclei are shown in blue. Scale bars, 10 μm. White arrows indicate the co-localization of 4-HNE in NOX4-positive astrocytes. (E) Quantification of NOX4 and 4-HNE positive astrocytes from immunofluorescence images in the cerebral cortex region from patients with AD (AD) or non-AD (normal) (n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2 The levels of oxidative stress-induced lipid peroxidation are elevated in impaired astrocytes of the cortex region from patients with Alzheimer's diseases. (A) Representative immunofluorescence images of 4-HNE protein expression in cerebral cortex region from patients with AD (AD #1, AD #2, AD #3) or non-AD (normal) showing 4-HNE (green) in astrocytes expressing astrocytes marker GFAP (red) around molecular layer (ML) (n = 3 per group, n = 10 images per individual subject). DAPI-stained nuclei are shown in blue. OS, Outer surface; ML, Molecular layer; EGL, External granular layer. Scale bars, 20 μm. White arrows indicate 4-HNE and GFAP positive cells. Symbols, which are expressed by white dotted line, indicate the distinct area among OS, ML, and EGL. (B) Quantification of intensity of 4-HNE positive staining in astrocytes from immunofluorescence images in the cerebral cortex region from patients with AD (AD #1, AD #2, AD #3) or non-AD (normal) (n = 3 per group, n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (C) Quantification of 4-HNE positive astrocytes from immunofluorescence images in the cerebral cortex region from patients with AD (AD #1, AD #2, AD #3) or non-AD (normal) (n = 3 per group, n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (D) Representative immunofluorescence images of 4-HNE protein expression in NOX4-positive astrocytes of patients with AD (AD) or non-AD (normal) showing 4-HNE (purple) in NOX4 (green)-positive astrocytes expressing astrocytes marker GFAP (red) around molecular layer (ML) (n = 10 images per individual subject). DAPI-stained nuclei are shown in blue. Scale bars, 10 μm. White arrows indicate the co-localization of 4-HNE in NOX4-positive astrocytes. (E) Quantification of NOX4 and 4-HNE positive astrocytes from immunofluorescence images in the cerebral cortex region from patients with AD (AD) or non-AD (normal) (n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)CC BY-NC-ND · PubMed Central
Fig. 3 The levels of NOX4 are elevated in impaired astrocytes of the cortex region from brain of APP/PS1 mice. (A) Representative immunofluorescence images of NOX4 protein expression in cortex region from brain of APP/PS1 mice (APP/PS1) or wild-type mice (WT) showing NOX4 (green) in astrocytes expressing astrocytes marker GFAP (red) (n = 5 per group, n = 10 images per individual subject). DAPI-stained nuclei are shown in blue. Scale bars, 20 μm. White arrows indicate NOX4 and GFAP positive cells. (B) Quantification of intensity for NOX4 positive staining in astrocytes from immunofluorescence images in the cortex region from brains of APP/PS1 mice (APP/PS1) or wild-type mice (WT) (n = 5 per group, n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (C) Quantification of NOX4 positive astrocytes from immunofluorescence images in the cortex region from brains of APP/PS1 mice (APP/PS1) or wild-type mice (WT) (n = 5 per group, n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3 The levels of NOX4 are elevated in impaired astrocytes of the cortex region from brain of APP/PS1 mice. (A) Representative immunofluorescence images of NOX4 protein expression in cortex region from brain of APP/PS1 mice (APP/PS1) or wild-type mice (WT) showing NOX4 (green) in astrocytes expressing astrocytes marker GFAP (red) (n = 5 per group, n = 10 images per individual subject). DAPI-stained nuclei are shown in blue. Scale bars, 20 μm. White arrows indicate NOX4 and GFAP positive cells. (B) Quantification of intensity for NOX4 positive staining in astrocytes from immunofluorescence images in the cortex region from brains of APP/PS1 mice (APP/PS1) or wild-type mice (WT) (n = 5 per group, n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (C) Quantification of NOX4 positive astrocytes from immunofluorescence images in the cortex region from brains of APP/PS1 mice (APP/PS1) or wild-type mice (WT) (n = 5 per group, n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)CC BY-NC-ND · PubMed Central
Fig. 4 The levels of oxidative stress-induced lipid peroxidation are elevated in impaired astrocytes of the cortex region from brains of APP/PS1 mice. (A) Representative immunofluorescence images of 4-HNE protein expression in cortex region from brain of APP/PS1 mice (APP/PS1) or wild-type mice (WT) showing 4-HNE (green) in astrocytes expressing astrocytes marker GFAP (red) (n = 5 per group, n = 10 images per individual subject). DAPI-stained nuclei are shown in blue. Scale bars, 20 μm. White arrows indicate 4-HNE and GFAP positive cells. (B) Quantification of 4-HNE positive staining in astrocytes from immunofluorescence images in the cortex region from brains of APP/PS1 mice (APP/PS1) or wild-type mice (WT) (n = 5 per group, n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (C) Quantification of 4-HNE positive astrocytes from immunofluorescence images of the cortex region from brains of APP/PS1 mice (APP/PS1) or wild-type mice (WT) (n = 5 per group, n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (D) Representative immunofluorescence images of 4-HNE protein expression in NOX4-positive astrocytes of APP/PS1 mice (APP/PS1) or wild-type mice (WT) showing 4-HNE (purple) in NOX4 (green)-positive astrocytes expressing astrocytes marker GFAP (red) (n = 10 images per individual subject). DAPI-stained nuclei are shown in blue. Scale bars, 10 μm. White arrows indicate the co-localization of 4-HNE in NOX4-positive astrocytes. (E) Quantification of NOX4 and 4-HNE positive astrocytes from immunofluorescence images in the cortex region from APP/PS1 mice (APP/PS1) or wild-type mice (WT) (n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4 The levels of oxidative stress-induced lipid peroxidation are elevated in impaired astrocytes of the cortex region from brains of APP/PS1 mice. (A) Representative immunofluorescence images of 4-HNE protein expression in cortex region from brain of APP/PS1 mice (APP/PS1) or wild-type mice (WT) showing 4-HNE (green) in astrocytes expressing astrocytes marker GFAP (red) (n = 5 per group, n = 10 images per individual subject). DAPI-stained nuclei are shown in blue. Scale bars, 20 μm. White arrows indicate 4-HNE and GFAP positive cells. (B) Quantification of 4-HNE positive staining in astrocytes from immunofluorescence images in the cortex region from brains of APP/PS1 mice (APP/PS1) or wild-type mice (WT) (n = 5 per group, n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (C) Quantification of 4-HNE positive astrocytes from immunofluorescence images of the cortex region from brains of APP/PS1 mice (APP/PS1) or wild-type mice (WT) (n = 5 per group, n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (D) Representative immunofluorescence images of 4-HNE protein expression in NOX4-positive astrocytes of APP/PS1 mice (APP/PS1) or wild-type mice (WT) showing 4-HNE (purple) in NOX4 (green)-positive astrocytes expressing astrocytes marker GFAP (red) (n = 10 images per individual subject). DAPI-stained nuclei are shown in blue. Scale bars, 10 μm. White arrows indicate the co-localization of 4-HNE in NOX4-positive astrocytes. (E) Quantification of NOX4 and 4-HNE positive astrocytes from immunofluorescence images in the cortex region from APP/PS1 mice (APP/PS1) or wild-type mice (WT) (n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)CC BY-NC-ND · PubMed Central
Fig. 5 The elevation of NOX4 promotes oxidative stress by impairment of mitochondrial metabolism via inhibition of mitochondrial respiration and ATP production in human astrocytes. (A) The levels of oxygen consumption rate (OCR) as the parameter of mitochondrial respiration activity and (B) quantification of OCR levels in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. Data are representative of three independent experiments. Data are mean ± SEM. **p < 0.01; *p < 0.05 using two-tailed Student's t-test. (C) Representative immunoblot analysis for five mitochondrial ETC protein levels (left) including NDUFB8 for Complex I (C I (NDUFB8)), SDHB for Complex II (C II (SDHB)), UQCRC2 for Complex III (C III (UQCRC2)), MTCO1 for Complex IV (C IV (MTCO1)) and ATP5F1A for Complex V (C V (ATP5F1A)) in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. Quantification for protein levels of C I (NDUFB8, C II (SDHB), C III (UQCRC2), C IV (MTCO1) and C V (ATP5F1A) (right) in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. For immunoblots, β-actin was used as a loading control. Data are representatives of three independent experiments. Data are mean ± standard deviation (SD). **p < 0.01; *p < 0.05 using two-tailed Student's t-test. (D) Quantification of mitochondrial ATP production rate in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. Data are mean ± SD. **p < 0.01 using two-tailed Student's t-test. (E) Quantification of mtROS levels using MitoSOX staining in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. Data are mean ± SD. *p < 0.05 using two-tailed Student's t-test. (F) Representative immunofluorescence images of mitochondrial morphology for mitochondria fragmentation by Tomm20 staining in control (Control) and NOX4 overexpressing (NOX4) human astrocytes showing Tomm20 (green) (n = 10 per group). DAPI-stained nuclei are shown in blue. The fragmentation of mitochondria is indicated (white arrows). Scale bars, 20 μm. Magnified views of the selected regions (upper right); scale bars, 5 μm. (G) Quantification of cells with mitochondrial fragmentation from immunofluorescence images of mitochondrial morphology in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group). (The percent of morphological dead cells in a total of 100 cells of 10 individual images per group was calculated). Symbols expressed by white dotted line indicate the shape of cells. Data are mean ± SD. **, p < 0.01 by Student's two-tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 5 The elevation of NOX4 promotes oxidative stress by impairment of mitochondrial metabolism via inhibition of mitochondrial respiration and ATP production in human astrocytes. (A) The levels of oxygen consumption rate (OCR) as the parameter of mitochondrial respiration activity and (B) quantification of OCR levels in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. Data are representative of three independent experiments. Data are mean ± SEM. **p < 0.01; *p < 0.05 using two-tailed Student's t-test. (C) Representative immunoblot analysis for five mitochondrial ETC protein levels (left) including NDUFB8 for Complex I (C I (NDUFB8)), SDHB for Complex II (C II (SDHB)), UQCRC2 for Complex III (C III (UQCRC2)), MTCO1 for Complex IV (C IV (MTCO1)) and ATP5F1A for Complex V (C V (ATP5F1A)) in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. Quantification for protein levels of C I (NDUFB8, C II (SDHB), C III (UQCRC2), C IV (MTCO1) and C V (ATP5F1A) (right) in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. For immunoblots, β-actin was used as a loading control. Data are representatives of three independent experiments. Data are mean ± standard deviation (SD). **p < 0.01; *p < 0.05 using two-tailed Student's t-test. (D) Quantification of mitochondrial ATP production rate in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. Data are mean ± SD. **p < 0.01 using two-tailed Student's t-test. (E) Quantification of mtROS levels using MitoSOX staining in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. Data are mean ± SD. *p < 0.05 using two-tailed Student's t-test. (F) Representative immunofluorescence images of mitochondrial morphology for mitochondria fragmentation by Tomm20 staining in control (Control) and NOX4 overexpressing (NOX4) human astrocytes showing Tomm20 (green) (n = 10 per group). DAPI-stained nuclei are shown in blue. The fragmentation of mitochondria is indicated (white arrows). Scale bars, 20 μm. Magnified views of the selected regions (upper right); scale bars, 5 μm. (G) Quantification of cells with mitochondrial fragmentation from immunofluorescence images of mitochondrial morphology in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group). (The percent of morphological dead cells in a total of 100 cells of 10 individual images per group was calculated). Symbols expressed by white dotted line indicate the shape of cells. Data are mean ± SD. **, p < 0.01 by Student's two-tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)CC BY-NC-ND · PubMed Central
Fig. 6 NOX4-induced mitochondrial metabolic impairment induces oxidative stress by inhibition of cellular antioxidant process in human astrocytes. (A-C) Quantification of (A) reduced GSH levels, (B) ratio of GSH2/GSSG, and (C) GSSG levels in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group). Data are mean ± SD. *, p < 0.01 by Student's two-tailed t-test. (D) Representative immunoblot analysis for nuclear and cytosolic NRF2 in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. For immunoblots, Histone H3 (nuclear) and β-actin (cytosolic) was used as a loading control. Data are representative of three independent experiments. Data are mean ± SD. **, p < 0.01; *, p < 0.05 using the two-tailed Student's t-test. (E–F) Quantification of (E) HO-1 protein levels, (F) HO-1 activity, (G) GCLC protein levels and (F) GCL activity in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group). Data are mean ± SD. *, p < 0.01 by Student's two-tailed t-test.
Fig. 6 NOX4-induced mitochondrial metabolic impairment induces oxidative stress by inhibition of cellular antioxidant process in human astrocytes. (A-C) Quantification of (A) reduced GSH levels, (B) ratio of GSH2/GSSG, and (C) GSSG levels in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group). Data are mean ± SD. *, p < 0.01 by Student's two-tailed t-test. (D) Representative immunoblot analysis for nuclear and cytosolic NRF2 in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. For immunoblots, Histone H3 (nuclear) and β-actin (cytosolic) was used as a loading control. Data are representative of three independent experiments. Data are mean ± SD. **, p < 0.01; *, p < 0.05 using the two-tailed Student's t-test. (E–F) Quantification of (E) HO-1 protein levels, (F) HO-1 activity, (G) GCLC protein levels and (F) GCL activity in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group). Data are mean ± SD. *, p < 0.01 by Student's two-tailed t-test.CC BY-NC-ND · PubMed Central
Fig. 7 NOX4 promotes ferroptosis by oxidative stress-induced lipid peroxidation in human astrocytes. (A) Representative immunofluorescence images of 4-HNE expression in control (Control) and NOX4 overexpressing (NOX4) human astrocytes showing 4-HNE (red) (n = 10 per group). DAPI-stained nuclei are shown in blue. The shape of cells showed shrinkage and lipid peroxidation-derived droplets in the plasma membrane were indicated (white arrows). Symbols expressed by white dotted line indicate the shape of cells. Scale bars, 20 μm. (B) Quantification of 4-HNE positive astrocytes from immunofluorescence images in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group) (The percent of morphological dead cells in a total of 100 cells of 10 individual images per group was calculated). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (C) Representative immunoblot analysis for 4-HNE and MDA protein levels (left) and quantification for 4-HNE and MDA protein levels in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. For immunoblots, β-actin was used as a loading control. Data are representative of three independent experiments. Data are mean ± SD. *p < 0.05 using the two-tailed Student's t-test. (D) Quantification of iron levels in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group). Data are mean ± standard deviation (SD). *, p < 0.05 by Student's two-tailed t-test. (E) Representative 3D images of control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 images per group). The morphological features of cytotoxicity were indicated (white arrows). Scale bars, 20 μm. (F) Quantification of the morphological dead cells in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group) (The percent of morphological dead cells in total 100 cells in 10 individual images per group). Data are mean ± SD. **, p < 0.01 using the two-tailed Student's t-test. (G) Cytotoxicity assay in control (Control) and NOX4 overexpressing (NOX4) human astrocytes was determined by lactate dehydrogenase (LDH) levels. Data are representatives of three independent experiments. Each experiment was done in triplicate. Data are mean ± SD. **, p < 0.01 using two-tailed Student's t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 7 NOX4 promotes ferroptosis by oxidative stress-induced lipid peroxidation in human astrocytes. (A) Representative immunofluorescence images of 4-HNE expression in control (Control) and NOX4 overexpressing (NOX4) human astrocytes showing 4-HNE (red) (n = 10 per group). DAPI-stained nuclei are shown in blue. The shape of cells showed shrinkage and lipid peroxidation-derived droplets in the plasma membrane were indicated (white arrows). Symbols expressed by white dotted line indicate the shape of cells. Scale bars, 20 μm. (B) Quantification of 4-HNE positive astrocytes from immunofluorescence images in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group) (The percent of morphological dead cells in a total of 100 cells of 10 individual images per group was calculated). Data are mean ± standard deviation (SD). **, p < 0.01 by Student's two-tailed t-test. (C) Representative immunoblot analysis for 4-HNE and MDA protein levels (left) and quantification for 4-HNE and MDA protein levels in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. For immunoblots, β-actin was used as a loading control. Data are representative of three independent experiments. Data are mean ± SD. *p < 0.05 using the two-tailed Student's t-test. (D) Quantification of iron levels in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group). Data are mean ± standard deviation (SD). *, p < 0.05 by Student's two-tailed t-test. (E) Representative 3D images of control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 images per group). The morphological features of cytotoxicity were indicated (white arrows). Scale bars, 20 μm. (F) Quantification of the morphological dead cells in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group) (The percent of morphological dead cells in total 100 cells in 10 individual images per group). Data are mean ± SD. **, p < 0.01 using the two-tailed Student's t-test. (G) Cytotoxicity assay in control (Control) and NOX4 overexpressing (NOX4) human astrocytes was determined by lactate dehydrogenase (LDH) levels. Data are representatives of three independent experiments. Each experiment was done in triplicate. Data are mean ± SD. **, p < 0.01 using two-tailed Student's t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)CC BY-NC-ND · PubMed Central
Fig. 8 A schematic diagram to summarize our new findings. Red arrow means an increase. Blue arrow means a decrease in the diagram. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 8 A schematic diagram to summarize our new findings. Red arrow means an increase. Blue arrow means a decrease in the diagram. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)CC BY-NC-ND · PubMed Central

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Claims the paper makes

58 Own result · 40 Not checked yet · 20 Own interpretation · 4 Own method

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  • It may state two separate claims as one. 5
  • Probably not a substantial claim. 35
37 of 37

  • The paper relies on this without support.

    The mechanisms by which NOX4 regulates ferroptosis of astrocytes by oxidative stress via impairment of mitochondrial metabolism in AD remain unclear.

    “However, the mechanisms by which NOX4 regulates ferroptosis of astrocytes by oxidative stress via impairment of mitochondrial metabolism in AD remains unclear.”

  • The paper relies on this without support.

    4-HNE is a marker for ferroptosis.

    “Since the levels of 4-HNE, a marker for ferroptosis, were elevated in impaired astrocytes of patients with AD”

  • The paper relies on this without support.

    Iron accumulation is a specific marker of ferroptosis.

    “the accumulation of iron, a specific marker of ferroptosis”

  • The paper relies on this without support.

    APP/PS1 mice model AD.

    “APP/PS1 double-transgenic mouse model of AD”

  • The paper relies on this without support.

    NRF2 is a key regulator of the cellular antioxidant response.

    “nuclear factor erythroid 2–related factor 2 (NRF2) signaling pathway, a key regulator of the cellular antioxidant response”

  • The paper relies on this without support.

    HO-1 and GCL are target genes of NRF2.

    “heme oxygenase-1 (HO-1) (Fig. 6E and F) and glutamate cysteine ligase (GCL) (Fig. 6E and F) as target genes of NRF2”

  • The paper relies on this without support.

    Mitochondrial fragmentation indicates mitochondrial impairment.

    “We next analyzed morphological impairment of mitochondria caused by NOX4 over-expression through immunofluorescence staining with Tomm20”

  • The paper relies on this without support.

    The upstream molecular target in the regulation of ferroptosis by lipid peroxidation in Alzheimer's disease remains unclear.

    “the upstream molecular target in the regulation of ferroptosis by lipid peroxidation in AD remains unclear”

  • The paper relies on this without support.

    NOX4 levels are high in the cortex of patients with Alzheimer's disease.

    “the high levels of NOX4, in cortex of patients with AD”

  • The paper relies on this without support.

    It is unclear whether impaired astrocytes in Alzheimer's disease have a function of reactive astrocytes.

    “it was unclear whether these impaired astrocytes have a function of reactive astrocytes in AD”
10 of 10

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4 of 4

Citations

Cites

Research works printed but not linked: 1

  1. Stockwell, 2017, Ferroptosis: a regulated cell death Nexus linking metabolism, Redox Biol. Dis. Cell, 171, 273

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Metadata

Authors
Park, M. W., Cha, H. W., Kim, J., Kim, J. H., Yang, H., Yoon, S., Boonpraman, N., Yi, S. S., Yoo, I. D., & Moon, J.-S.
Affiliations
  • Soonchunhyang University (KR)
  • Soonchunhyang University Hospital Cheonan (KR)
Corresponding author
  • Yoo, Ik Dong
  • Moon, Jong-Seok
ORCID
Venue
Redox Biology
Publisher
Elsevier BV
Published
2021-05
Volume
41
Pages
101947
ISSN
2213-2317 (ISSN-L)
DOI
10.1016/j.redox.2021.101947
PMC
PMC8027773
OpenAlex
W3136661179
Cited by
683
References
92
Citations by year
2026: 139 · 2025: 145 · 2024: 187 · 2023: 128 · 2022: 74 · 2021: 10
FWCI
62.90
Type
journal article
Language
en
Open access
Licence
Locations
  • Redox Biology (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Soonchunhyang University
  • National Research Foundation of Korea
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
Keywords
NOX4, ferroptosis, astrocytes, Alzheimer's disease, oxidative stress, lipid peroxidation, mitochondrial dysfunction, mitochondrial respiration, mitochondrial electron transport chain, mitochondrial reactive oxygen species, mitochondrial fragmentation, 4-hydroxynonenal, malondialdehyde, APP/PS1 mice, reactive oxygen species
MeSH terms
NADPH Oxidase 4, Ferroptosis, Alzheimer Disease, Animals, Astrocytes, Humans, Mitochondria, Lipid Peroxidation, Reactive Oxygen Species, Oxidative Stress, Mice

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