Hypoxia causes autophagic stress and derangement of metabolic adaptation in a cell model of amyotrophic lateral sclerosis

Authors

  • Sara Cimini,

    1. Laboratory of Molecular Pathology, Department of Molecular Biochemistry and Pharmacology, IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy
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    • These authors contributed equally to this work.
  • Milena Rizzardini,

    1. Laboratory of Molecular Pathology, Department of Molecular Biochemistry and Pharmacology, IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy
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    • These authors contributed equally to this work.
  • Gloria Biella,

    1. Unit of Genetics of Neurodegenerative Disorders, Department of Neuroscience, IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy
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  • Lavinia Cantoni

    Corresponding author
    1. Laboratory of Molecular Pathology, Department of Molecular Biochemistry and Pharmacology, IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy
    • Address correspondence and reprint requests to Lavinia Cantoni, Laboratory of Molecular Pathology, IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, Via G. La Masa 19, 20156 Milan, Italy.

      E-mail: lavinia.cantoni@marionegri.it

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Abstract

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease that affects motor neurons. The recruitment of autophagy (macroautophagy) and mitochondrial dysfunction are documented in amyotrophic lateral sclerosis patients and experimental models expressing mutant forms of Cu, Zn superoxide dismutase (SOD1) protein, but their impact in the disease remains unclear. Hypoxia is a stress closely related to the disease in patients and mutant SOD1 mice; in individual cells, hypoxia activates autophagy and regulates mitochondrial metabolism as fundamental adaptive mechanisms. Our aim was to examine whether mutant SOD1 changed this response. Hypoxia (1% O2 for 22 h) caused greater loss of viability and more marked activation of caspase 3/7 in the motor neuronal NSC-34 cell line stably transfected with the G93A mutant human SOD1 (G93A-NSC) than in the one with the wild-type SOD1 (WT-NSC) or in untransfected NSC-34. In the G93A-NSC cells, there was a more marked accumulation of the LC3-II autophagy protein, attributable to autophagic stress; 3-methyladenine, which acts on initiation of autophagy, fully rescued G93A-NSC viability and reduced the activation of caspase 3/7 indicating this was a secondary event; the metabolic handling of hypoxia was inappropriate possibly contributing to the autophagic stress. Our findings evidentiate that the G93A mutation of SOD1 profoundly altered the adaptive metabolic response to hypoxia and this could increase the cell susceptibility to this stress.

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Hypoxia activates autophagy and modifies glycolysis and mitochondrial respiration as fundamental cell adaptive mechanisms. This stress is closely related to amyotrophic lateral sclerosis. The recruitment of autophagy and mitochondrial dysfunction are documented in patients and models expressing mutant Cu, Zn superoxide dismutase (SOD1) protein, but their impact in the disease remains unclear. G93ASOD1 cells were more susceptible to hypoxia than wild-type SOD1 cells and showed autophagic stress and inappropriate handling of energy metabolism. Defective adaptation to hypoxia may contribute to neurodegeneration.

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