Mitochondrial-localized NADPH oxidase 4 is a source of superoxide in angiotensin II-stimulated neurons

Adam Case, Shumin Li, Urmi Basu, Jun Tian, Matthew C Zimmerman

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Abstract

Angiotensin II (ANG II) plays an important role in the central regulation of systemic cardiovascular function. ANG II-mediated intraneuronal signaling has been shown to be predicated by an increase in mitochondrial superoxide (O2̇-), yet the source of this reactive oxygen species (ROS) production remains unclear. NADPH oxidase 4 (Nox4), a member of the NADPH oxidase family, has been reported to be localized in mitochondria of various cell types and has been implicated in brain angiotensinergic signaling. However, the subcellular localization and function of Nox4 in neurons has not been fully elucidated. In this study, we hypothesized that Nox4 is expressed in neuron mitochondria and is involved in ANG II-dependent O2̇--mediated intraneuronal signaling. To query this, Nox4 immunofluorescent staining and mitochondrial enrichment were performed in a mouse catecholaminergic neuronal cell model (CATH.a). Nox4 was shown to be present in neuron mitochondria as evidenced by colocalization with both the mitochondrial-localized protein manganese superoxide dismutase (MnSOD) and dye MitoTracker Red. Moreover, Nox4 expression was significantly increased in enriched mitochondrial fractions compared with whole cell lysates. Additionally, adenoviral-encoded small interfering RNA for Nox4 (AdsiNox4) caused a robust knockdown in Nox4 mRNA and protein levels, which led to the attenuation of ANG II-induced mitochondrial O2̇- production. Finally, in the subfornical organ (SFO) of the brain, Nox4 not only demonstrated mitochondrial localization but was induced by chronic, peripheral infusion of ANG II. Collectively, these data suggest that Nox4 is a source of O2̇- in neuron mitochondria that contributes to ANG II intraneuronal signaling.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume305
Issue number1
DOIs
StatePublished - Jul 1 2013

Fingerprint

NADPH Oxidase
Superoxides
Angiotensin II
Neurons
Mitochondria
Subfornical Organ
Mitochondrial Proteins
Brain
Small Interfering RNA
Superoxide Dismutase
Reactive Oxygen Species
Coloring Agents
Staining and Labeling

Keywords

  • Angiotensin II
  • CATH.a neurons
  • Mitochondria
  • Nox4
  • Subfornical organ
  • siRNA

ASJC Scopus subject areas

  • Physiology
  • Physiology (medical)
  • Cardiology and Cardiovascular Medicine

Cite this

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title = "Mitochondrial-localized NADPH oxidase 4 is a source of superoxide in angiotensin II-stimulated neurons",
abstract = "Angiotensin II (ANG II) plays an important role in the central regulation of systemic cardiovascular function. ANG II-mediated intraneuronal signaling has been shown to be predicated by an increase in mitochondrial superoxide (O2̇-), yet the source of this reactive oxygen species (ROS) production remains unclear. NADPH oxidase 4 (Nox4), a member of the NADPH oxidase family, has been reported to be localized in mitochondria of various cell types and has been implicated in brain angiotensinergic signaling. However, the subcellular localization and function of Nox4 in neurons has not been fully elucidated. In this study, we hypothesized that Nox4 is expressed in neuron mitochondria and is involved in ANG II-dependent O2̇--mediated intraneuronal signaling. To query this, Nox4 immunofluorescent staining and mitochondrial enrichment were performed in a mouse catecholaminergic neuronal cell model (CATH.a). Nox4 was shown to be present in neuron mitochondria as evidenced by colocalization with both the mitochondrial-localized protein manganese superoxide dismutase (MnSOD) and dye MitoTracker Red. Moreover, Nox4 expression was significantly increased in enriched mitochondrial fractions compared with whole cell lysates. Additionally, adenoviral-encoded small interfering RNA for Nox4 (AdsiNox4) caused a robust knockdown in Nox4 mRNA and protein levels, which led to the attenuation of ANG II-induced mitochondrial O2̇- production. Finally, in the subfornical organ (SFO) of the brain, Nox4 not only demonstrated mitochondrial localization but was induced by chronic, peripheral infusion of ANG II. Collectively, these data suggest that Nox4 is a source of O2̇- in neuron mitochondria that contributes to ANG II intraneuronal signaling.",
keywords = "Angiotensin II, CATH.a neurons, Mitochondria, Nox4, Subfornical organ, siRNA",
author = "Adam Case and Shumin Li and Urmi Basu and Jun Tian and Zimmerman, {Matthew C}",
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T1 - Mitochondrial-localized NADPH oxidase 4 is a source of superoxide in angiotensin II-stimulated neurons

AU - Case, Adam

AU - Li, Shumin

AU - Basu, Urmi

AU - Tian, Jun

AU - Zimmerman, Matthew C

PY - 2013/7/1

Y1 - 2013/7/1

N2 - Angiotensin II (ANG II) plays an important role in the central regulation of systemic cardiovascular function. ANG II-mediated intraneuronal signaling has been shown to be predicated by an increase in mitochondrial superoxide (O2̇-), yet the source of this reactive oxygen species (ROS) production remains unclear. NADPH oxidase 4 (Nox4), a member of the NADPH oxidase family, has been reported to be localized in mitochondria of various cell types and has been implicated in brain angiotensinergic signaling. However, the subcellular localization and function of Nox4 in neurons has not been fully elucidated. In this study, we hypothesized that Nox4 is expressed in neuron mitochondria and is involved in ANG II-dependent O2̇--mediated intraneuronal signaling. To query this, Nox4 immunofluorescent staining and mitochondrial enrichment were performed in a mouse catecholaminergic neuronal cell model (CATH.a). Nox4 was shown to be present in neuron mitochondria as evidenced by colocalization with both the mitochondrial-localized protein manganese superoxide dismutase (MnSOD) and dye MitoTracker Red. Moreover, Nox4 expression was significantly increased in enriched mitochondrial fractions compared with whole cell lysates. Additionally, adenoviral-encoded small interfering RNA for Nox4 (AdsiNox4) caused a robust knockdown in Nox4 mRNA and protein levels, which led to the attenuation of ANG II-induced mitochondrial O2̇- production. Finally, in the subfornical organ (SFO) of the brain, Nox4 not only demonstrated mitochondrial localization but was induced by chronic, peripheral infusion of ANG II. Collectively, these data suggest that Nox4 is a source of O2̇- in neuron mitochondria that contributes to ANG II intraneuronal signaling.

AB - Angiotensin II (ANG II) plays an important role in the central regulation of systemic cardiovascular function. ANG II-mediated intraneuronal signaling has been shown to be predicated by an increase in mitochondrial superoxide (O2̇-), yet the source of this reactive oxygen species (ROS) production remains unclear. NADPH oxidase 4 (Nox4), a member of the NADPH oxidase family, has been reported to be localized in mitochondria of various cell types and has been implicated in brain angiotensinergic signaling. However, the subcellular localization and function of Nox4 in neurons has not been fully elucidated. In this study, we hypothesized that Nox4 is expressed in neuron mitochondria and is involved in ANG II-dependent O2̇--mediated intraneuronal signaling. To query this, Nox4 immunofluorescent staining and mitochondrial enrichment were performed in a mouse catecholaminergic neuronal cell model (CATH.a). Nox4 was shown to be present in neuron mitochondria as evidenced by colocalization with both the mitochondrial-localized protein manganese superoxide dismutase (MnSOD) and dye MitoTracker Red. Moreover, Nox4 expression was significantly increased in enriched mitochondrial fractions compared with whole cell lysates. Additionally, adenoviral-encoded small interfering RNA for Nox4 (AdsiNox4) caused a robust knockdown in Nox4 mRNA and protein levels, which led to the attenuation of ANG II-induced mitochondrial O2̇- production. Finally, in the subfornical organ (SFO) of the brain, Nox4 not only demonstrated mitochondrial localization but was induced by chronic, peripheral infusion of ANG II. Collectively, these data suggest that Nox4 is a source of O2̇- in neuron mitochondria that contributes to ANG II intraneuronal signaling.

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KW - Mitochondria

KW - Nox4

KW - Subfornical organ

KW - siRNA

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JO - American Journal of Physiology - Renal Physiology

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