Transitory phases of autophagic death and programmed necrosis during superoxide-induced neuronal cell death

Gavin Cl Higgins, Rodney J Devenish, Philip M Beart, Phillip Nagley

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14 Citations (Scopus)

Abstract

Neurons can undergo a diverse range of death responses under oxidative stress, encompassing apoptosis (caspase-dependent, programmed cell death) to various forms of caspase-independent death, including necrosis. We recently showed that primary murine cortical neurons exposed acutely to hydrogen peroxide undergo caspase-independent death, both ACD and programmed necrosis. To determine how oxidative stress induced by superoxide affects the route to cellular demise, we exposed primary cortical neurons to extended superoxide insult (provided by exogenous xanthine and xanthine oxidase in the presence of catalase). Under these conditions, over 24 h, the nitroblue tetrazolium-reducing activity (indicative of superoxide) rose significantly during the first 4 to 8 h and then declined to background levels. As with hydrogen peroxide, this superoxide insult failed to activate downstream caspases (a??3, a??7, and a??9). Substantial depolarization of mitochondria occurred after 1 h, and nuclear morphology changes characteristic of oxidative stress became maximal after 2 h. However, death indicated by plasma membrane permeabilization (cellular uptake of propidium iodide) approached maximal levels only after 4 h, at which time substantial redistribution to the cytosol of death-associated mitochondrial intermembrane space proteins, notably endonuclease G, had occurred. Applying established criteria for autophagic death (knockdown of Atg7) or programmed necrosis (knockdown of endonuclease G), cells treated with the relevant siRNA showed significant blockade of each type of cell death, 4 h after onset of the superoxide flux. Yet at later times, siRNA-mediated knockdown failed to prevent death, monitored by cellular uptake of propidium iodide. We conclude that superoxide initially invokes a diverse programmed caspase-independent death response, involving transient manifestation in parallel of autophagic death and programmed necrosis. Ultimately most neurons become overwhelmed by the consequences of severe oxidative stress and die. This study reveals the multiple phases of neuronal cell death modalities under extended oxidative stress.
Original languageEnglish
Pages (from-to)1960 - 1967
Number of pages8
JournalFree Radical Biology and Medicine
Volume53
Issue number10
DOIs
Publication statusPublished - 2012

Cite this

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title = "Transitory phases of autophagic death and programmed necrosis during superoxide-induced neuronal cell death",
abstract = "Neurons can undergo a diverse range of death responses under oxidative stress, encompassing apoptosis (caspase-dependent, programmed cell death) to various forms of caspase-independent death, including necrosis. We recently showed that primary murine cortical neurons exposed acutely to hydrogen peroxide undergo caspase-independent death, both ACD and programmed necrosis. To determine how oxidative stress induced by superoxide affects the route to cellular demise, we exposed primary cortical neurons to extended superoxide insult (provided by exogenous xanthine and xanthine oxidase in the presence of catalase). Under these conditions, over 24 h, the nitroblue tetrazolium-reducing activity (indicative of superoxide) rose significantly during the first 4 to 8 h and then declined to background levels. As with hydrogen peroxide, this superoxide insult failed to activate downstream caspases (a??3, a??7, and a??9). Substantial depolarization of mitochondria occurred after 1 h, and nuclear morphology changes characteristic of oxidative stress became maximal after 2 h. However, death indicated by plasma membrane permeabilization (cellular uptake of propidium iodide) approached maximal levels only after 4 h, at which time substantial redistribution to the cytosol of death-associated mitochondrial intermembrane space proteins, notably endonuclease G, had occurred. Applying established criteria for autophagic death (knockdown of Atg7) or programmed necrosis (knockdown of endonuclease G), cells treated with the relevant siRNA showed significant blockade of each type of cell death, 4 h after onset of the superoxide flux. Yet at later times, siRNA-mediated knockdown failed to prevent death, monitored by cellular uptake of propidium iodide. We conclude that superoxide initially invokes a diverse programmed caspase-independent death response, involving transient manifestation in parallel of autophagic death and programmed necrosis. Ultimately most neurons become overwhelmed by the consequences of severe oxidative stress and die. This study reveals the multiple phases of neuronal cell death modalities under extended oxidative stress.",
author = "Higgins, {Gavin Cl} and Devenish, {Rodney J} and Beart, {Philip M} and Phillip Nagley",
year = "2012",
doi = "10.1016/j.freeradbiomed.2012.08.586",
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journal = "Free Radical Biology and Medicine",
issn = "0891-5849",
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Transitory phases of autophagic death and programmed necrosis during superoxide-induced neuronal cell death. / Higgins, Gavin Cl; Devenish, Rodney J; Beart, Philip M; Nagley, Phillip.

In: Free Radical Biology and Medicine, Vol. 53, No. 10, 2012, p. 1960 - 1967.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Transitory phases of autophagic death and programmed necrosis during superoxide-induced neuronal cell death

AU - Higgins, Gavin Cl

AU - Devenish, Rodney J

AU - Beart, Philip M

AU - Nagley, Phillip

PY - 2012

Y1 - 2012

N2 - Neurons can undergo a diverse range of death responses under oxidative stress, encompassing apoptosis (caspase-dependent, programmed cell death) to various forms of caspase-independent death, including necrosis. We recently showed that primary murine cortical neurons exposed acutely to hydrogen peroxide undergo caspase-independent death, both ACD and programmed necrosis. To determine how oxidative stress induced by superoxide affects the route to cellular demise, we exposed primary cortical neurons to extended superoxide insult (provided by exogenous xanthine and xanthine oxidase in the presence of catalase). Under these conditions, over 24 h, the nitroblue tetrazolium-reducing activity (indicative of superoxide) rose significantly during the first 4 to 8 h and then declined to background levels. As with hydrogen peroxide, this superoxide insult failed to activate downstream caspases (a??3, a??7, and a??9). Substantial depolarization of mitochondria occurred after 1 h, and nuclear morphology changes characteristic of oxidative stress became maximal after 2 h. However, death indicated by plasma membrane permeabilization (cellular uptake of propidium iodide) approached maximal levels only after 4 h, at which time substantial redistribution to the cytosol of death-associated mitochondrial intermembrane space proteins, notably endonuclease G, had occurred. Applying established criteria for autophagic death (knockdown of Atg7) or programmed necrosis (knockdown of endonuclease G), cells treated with the relevant siRNA showed significant blockade of each type of cell death, 4 h after onset of the superoxide flux. Yet at later times, siRNA-mediated knockdown failed to prevent death, monitored by cellular uptake of propidium iodide. We conclude that superoxide initially invokes a diverse programmed caspase-independent death response, involving transient manifestation in parallel of autophagic death and programmed necrosis. Ultimately most neurons become overwhelmed by the consequences of severe oxidative stress and die. This study reveals the multiple phases of neuronal cell death modalities under extended oxidative stress.

AB - Neurons can undergo a diverse range of death responses under oxidative stress, encompassing apoptosis (caspase-dependent, programmed cell death) to various forms of caspase-independent death, including necrosis. We recently showed that primary murine cortical neurons exposed acutely to hydrogen peroxide undergo caspase-independent death, both ACD and programmed necrosis. To determine how oxidative stress induced by superoxide affects the route to cellular demise, we exposed primary cortical neurons to extended superoxide insult (provided by exogenous xanthine and xanthine oxidase in the presence of catalase). Under these conditions, over 24 h, the nitroblue tetrazolium-reducing activity (indicative of superoxide) rose significantly during the first 4 to 8 h and then declined to background levels. As with hydrogen peroxide, this superoxide insult failed to activate downstream caspases (a??3, a??7, and a??9). Substantial depolarization of mitochondria occurred after 1 h, and nuclear morphology changes characteristic of oxidative stress became maximal after 2 h. However, death indicated by plasma membrane permeabilization (cellular uptake of propidium iodide) approached maximal levels only after 4 h, at which time substantial redistribution to the cytosol of death-associated mitochondrial intermembrane space proteins, notably endonuclease G, had occurred. Applying established criteria for autophagic death (knockdown of Atg7) or programmed necrosis (knockdown of endonuclease G), cells treated with the relevant siRNA showed significant blockade of each type of cell death, 4 h after onset of the superoxide flux. Yet at later times, siRNA-mediated knockdown failed to prevent death, monitored by cellular uptake of propidium iodide. We conclude that superoxide initially invokes a diverse programmed caspase-independent death response, involving transient manifestation in parallel of autophagic death and programmed necrosis. Ultimately most neurons become overwhelmed by the consequences of severe oxidative stress and die. This study reveals the multiple phases of neuronal cell death modalities under extended oxidative stress.

UR - http://www.sciencedirect.com/science/article/pii/S0891584912011033

U2 - 10.1016/j.freeradbiomed.2012.08.586

DO - 10.1016/j.freeradbiomed.2012.08.586

M3 - Article

VL - 53

SP - 1960

EP - 1967

JO - Free Radical Biology and Medicine

JF - Free Radical Biology and Medicine

SN - 0891-5849

IS - 10

ER -