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Transcriptional block of AMPK-induced autophagy promotes glutamate excitotoxicity in nutrient-deprived SH-SY5Y neuroblastoma cells.
Cell Mol Life Sci. 2020 Sep; 77(17):3383-3399.CM

Abstract

We investigated the role of autophagy, a controlled lysosomal degradation of cellular macromolecules and organelles, in glutamate excitotoxicity during nutrient deprivation in vitro. The incubation in low-glucose serum/amino acid-free cell culture medium synergized with glutamate in increasing AMP/ATP ratio and causing excitotoxic necrosis in SH-SY5Y human neuroblastoma cells. Glutamate suppressed starvation-triggered autophagy, as confirmed by diminished intracellular acidification, lower LC3 punctuation and LC3-I conversion to autophagosome-associated LC3-II, reduced expression of proautophagic beclin-1 and ATG5, increase of the selective autophagic target NBR1, and decreased number of autophagic vesicles. Similar results were observed in PC12 rat pheochromocytoma cells. Both glutamate-mediated excitotoxicity and autophagy inhibition in starved SH-SY5Y cells were reverted by NMDA antagonist memantine and mimicked by NMDA agonists D-aspartate and ibotenate. Glutamate reduced starvation-triggered phosphorylation of the energy sensor AMP-activated protein kinase (AMPK) without affecting the activity of mammalian target of rapamycin complex 1, a major negative regulator of autophagy. This was associated with reduced mRNA levels of autophagy transcriptional activators (FOXO3, ATF4) and molecules involved in autophagy initiation (ULK1, ATG13, FIP200), autophagosome nucleation/elongation (ATG14, beclin-1, ATG5), and autophagic cargo delivery to autophagosomes (SQSTM1). Glutamate-mediated transcriptional repression of autophagy was alleviated by overexpression of constitutively active AMPK. Genetic or pharmacological AMPK activation by AMPK overexpression or metformin, as well as genetic or pharmacological autophagy induction by TFEB overexpression or lithium chloride, reduced the sensitivity of nutrient-deprived SH-SY5Y cells to glutamate excitotoxicity. These data indicate that transcriptional inhibition of AMPK-dependent cytoprotective autophagy is involved in glutamate-mediated excitotoxicity during nutrient deprivation in vitro.

Authors+Show Affiliations

Department of Neurophysiology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Belgrade, Serbia.Department of Neurophysiology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Belgrade, Serbia.Faculty of Medicine, Institute of Histology and Embryology, University of Belgrade, Belgrade, Serbia.Faculty of Medicine, Institute of Histology and Embryology, University of Belgrade, Belgrade, Serbia.Faculty of Medicine, Institute of Medical and Clinical Biochemistry, University of Belgrade, Belgrade, Serbia.Faculty of Medicine, Institute of Medical and Clinical Biochemistry, University of Belgrade, Belgrade, Serbia.Faculty of Medicine, Institute of Medical and Clinical Biochemistry, University of Belgrade, Belgrade, Serbia.Department of Neurobiology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Belgrade, Serbia.Inserm U1016, Institut Cochin, Paris, France. CNRS UMR8104, Paris, France. Université Paris Descartes, Sorbonne Paris cité, Paris, France.Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK. Regional Center for Biomedical Research, University of Castilla-La Mancha, Ciudad Real, Spain.Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK.Faculty of Medicine, Institute of Microbiology and Immunology, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia. vladimir.trajkovic@med.bg.ac.rs.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31720741

Citation

Vucicevic, Ljubica, et al. "Transcriptional Block of AMPK-induced Autophagy Promotes Glutamate Excitotoxicity in Nutrient-deprived SH-SY5Y Neuroblastoma Cells." Cellular and Molecular Life Sciences : CMLS, vol. 77, no. 17, 2020, pp. 3383-3399.
Vucicevic L, Misirkic M, Ciric D, et al. Transcriptional block of AMPK-induced autophagy promotes glutamate excitotoxicity in nutrient-deprived SH-SY5Y neuroblastoma cells. Cell Mol Life Sci. 2020;77(17):3383-3399.
Vucicevic, L., Misirkic, M., Ciric, D., Martinovic, T., Jovanovic, M., Isakovic, A., Markovic, I., Saponjic, J., Foretz, M., Rabanal-Ruiz, Y., Korolchuk, V. I., & Trajkovic, V. (2020). Transcriptional block of AMPK-induced autophagy promotes glutamate excitotoxicity in nutrient-deprived SH-SY5Y neuroblastoma cells. Cellular and Molecular Life Sciences : CMLS, 77(17), 3383-3399. https://doi.org/10.1007/s00018-019-03356-2
Vucicevic L, et al. Transcriptional Block of AMPK-induced Autophagy Promotes Glutamate Excitotoxicity in Nutrient-deprived SH-SY5Y Neuroblastoma Cells. Cell Mol Life Sci. 2020;77(17):3383-3399. PubMed PMID: 31720741.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Transcriptional block of AMPK-induced autophagy promotes glutamate excitotoxicity in nutrient-deprived SH-SY5Y neuroblastoma cells. AU - Vucicevic,Ljubica, AU - Misirkic,Maja, AU - Ciric,Darko, AU - Martinovic,Tamara, AU - Jovanovic,Maja, AU - Isakovic,Aleksandra, AU - Markovic,Ivanka, AU - Saponjic,Jasna, AU - Foretz,Marc, AU - Rabanal-Ruiz,Yoana, AU - Korolchuk,Viktor I, AU - Trajkovic,Vladimir, Y1 - 2019/11/12/ PY - 2019/05/20/received PY - 2019/10/28/accepted PY - 2019/10/16/revised PY - 2019/11/14/pubmed PY - 2020/8/22/medline PY - 2019/11/14/entrez KW - Brain KW - Energy stress KW - Ischemia KW - Neurodegeneration KW - Neurotoxicity SP - 3383 EP - 3399 JF - Cellular and molecular life sciences : CMLS JO - Cell Mol Life Sci VL - 77 IS - 17 N2 - We investigated the role of autophagy, a controlled lysosomal degradation of cellular macromolecules and organelles, in glutamate excitotoxicity during nutrient deprivation in vitro. The incubation in low-glucose serum/amino acid-free cell culture medium synergized with glutamate in increasing AMP/ATP ratio and causing excitotoxic necrosis in SH-SY5Y human neuroblastoma cells. Glutamate suppressed starvation-triggered autophagy, as confirmed by diminished intracellular acidification, lower LC3 punctuation and LC3-I conversion to autophagosome-associated LC3-II, reduced expression of proautophagic beclin-1 and ATG5, increase of the selective autophagic target NBR1, and decreased number of autophagic vesicles. Similar results were observed in PC12 rat pheochromocytoma cells. Both glutamate-mediated excitotoxicity and autophagy inhibition in starved SH-SY5Y cells were reverted by NMDA antagonist memantine and mimicked by NMDA agonists D-aspartate and ibotenate. Glutamate reduced starvation-triggered phosphorylation of the energy sensor AMP-activated protein kinase (AMPK) without affecting the activity of mammalian target of rapamycin complex 1, a major negative regulator of autophagy. This was associated with reduced mRNA levels of autophagy transcriptional activators (FOXO3, ATF4) and molecules involved in autophagy initiation (ULK1, ATG13, FIP200), autophagosome nucleation/elongation (ATG14, beclin-1, ATG5), and autophagic cargo delivery to autophagosomes (SQSTM1). Glutamate-mediated transcriptional repression of autophagy was alleviated by overexpression of constitutively active AMPK. Genetic or pharmacological AMPK activation by AMPK overexpression or metformin, as well as genetic or pharmacological autophagy induction by TFEB overexpression or lithium chloride, reduced the sensitivity of nutrient-deprived SH-SY5Y cells to glutamate excitotoxicity. These data indicate that transcriptional inhibition of AMPK-dependent cytoprotective autophagy is involved in glutamate-mediated excitotoxicity during nutrient deprivation in vitro. SN - 1420-9071 UR - https://www.unboundmedicine.com/medline/citation/31720741/Transcriptional_block_of_AMPK_induced_autophagy_promotes_glutamate_excitotoxicity_in_nutrient_deprived_SH_SY5Y_neuroblastoma_cells_ L2 - https://doi.org/10.1007/s00018-019-03356-2 DB - PRIME DP - Unbound Medicine ER -