EFFECT OF GLUTAMATE ON AUTOPHAGY INDUCED BY ENERGY STRESS IN SH-SY5Y NEURONS
Abstract
Abstract
Introduction: Autophagy is a cytoprotective mechanism by which cells adapt to stress. In neurons, the question whether enhanced autophagy promotes cell death or cell survival is a subject of considerable debate, especially in excitotoxic conditions such as cerebral ischemia. Excitotoxicity is a pathological process that involves overstimulation of the excitatory receptors, primarily the ionotropic and metabotropic glutamate receptors.
Material and methods: The viability of neuroblastoma SH-SY5Y cells was investigated by MTT assay for mitochondrial dehydrogenase activity. The appearance of autophagolysosomes and autophagic flux were determined by acridine orange staining (AO) and immunoblot, and the expression of autophagic genes was assessed by quantitative RT-PCR.
Results: Viability test showed a decrease in cell numbers, while AO test demonstrated a decrease in number of acidic vesicles in HBSS medium with glutamate compared to HBSS medium alone. Accumulation of autophagosome-asociated LC3-II protein was observed by immunoblot in HBSS medium, while LC3-II levels were lower in HBSS medium with glutamate. Quantitative RT-PCR showed reduced expression of the autophagy genes in HBSS medium with glutamate compared to the HBSS medium.
Conclusion: The research confirmed that glutamate during energy deprivation in vitro leads to a decrease in autophagy in neurons, possibly preventing the induction of autophagy by reducing the transcription of autophagy genes.
Keywords: autophagy, excitotoxicity, glutamate.
References
Mizushima N. Autophagy in protein and organelle turnover. Cold Spring Harbor symposia on quantitative biology 2011; 76:397-402.
Mizushima N, Klionsky DJ. Protein turnover via autophagy: implications for metabolism. Annual review of nutrition 2007; 27:19-40.
Xie Z, Klionsky DJ. Autophagosome formation: core machinery and adaptations. Nature Cell Biology 2007; 9:1102-9.
Tsukamoto S, Kuma A, Murakami M, Kishi C, Yamamoto A, Mizushima N. Autophagy is essential for preimplantation development of mouse embryos. Science 2008; 321:117-20.
Mari G, Niso Santano M, Baehrecke EH, Kroemer G. Self-consumption: the interplay of autophagy and apoptosis. Nature Reviews Molecular Cell Biology 2014; 15:81-94.
Mizushima N, Yoshimori T, Ohsumi Y. The role of Atg proteins in autophagosome formation. The Annual Review of Cell and Developmental Biology 2011; 27:107-32.
Kung CP, Budina A, Balaburski G, Bergenstock MK, Murphy M. Autophagy in tumor suppression and cancer therapy. Critical Reviews in Eukaryot Gene Expression 2011; 21:71-100.
Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, et al. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. The Journal of Cell Biology 2008; 182:685-701.
Puyal J, Ginet V, Clarke PGH. Multiple interacting cell death mechanisms in the mediation of excitotoxicity and ischemic brain damage. A challenge for neuroprotection. Progress in Neurobiology 2013; 105:24–48.
Wirawa E, Vanden Berghe T, Lippens S, Agostinis P, Vandenabeele P. Autophagy: for better or for worse. Cell Research 2012; 22:43-61.
Komatsu M, Waguri S, Koike M, Sou YS, Ueno T, Hara T, et al. Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell 2007; 131:1149-63.
Johansen T, Lamark T. Selective autophagy mediated by autophagic adapter proteins. Autophagy 2011; 7:279-96.
Kulbea JR, Mulcahy Levya JM, Coultrap SJ, Thorburn A, Ulrich Bayer K. Excitotoxic glutamate insults block autophagic flux in hippocampal neurons. Brain Research 2014; 1542:12–9.
Rubinsztein DC. The roles of intracellular protein-degradation pathways in neurodegeneration. Nature 2006; 443:780-6.
Scherz-Shouval R, Shvets E, Fass E, Shorer H, Gil L, Elazar Z. Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4. The EMBO Journal 2007; 26:1749-60.
Ginet V, Spiehlmann A, Rummel C, Rudinskiy N, Grishchuk Y, Luthi-Carter R, et al. Involvement of autophagy in hypoxic-excitotoxic neuronal death. Autophagy. Landes Bioscience 2014; 10:846–60.