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Int. J. Mol. Sci. 2014, 15(10), 19253-19264; doi:10.3390/ijms151019253

DRAM1 Protects Neuroblastoma Cells from Oxygen-Glucose Deprivation/Reperfusion-Induced Injury via Autophagy

Department of Neurosurgery, Second Xiangya Hospital of Central South University, Changsha 410011, China
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Received: 15 July 2014 / Revised: 18 September 2014 / Accepted: 9 October 2014 / Published: 23 October 2014
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Abstract

DNA damage-regulated autophagy modulator protein 1 (DRAM1), a multi-pass membrane lysosomal protein, is reportedly a tumor protein p53 (TP53) target gene involved in autophagy. During cerebral ischemia/reperfusion (I/R) injury, DRAM1 protein expression is increased, and autophagy is activated. However, the functional significance of DRAM1 and the relationship between DRAM1 and autophagy in brain I/R remains uncertain. The aim of this study is to investigate whether DRAM1 mediates autophagy activation in cerebral I/R injury and to explore its possible effects and mechanisms. We adopt the oxygen-glucose deprivation and reperfusion (OGD/R) Neuro-2a cell model to mimic cerebral I/R conditions in vitro, and RNA interference is used to knock down DRAM1 expression in this model. Cell viability assay is performed using the LIVE/DEAD viability/cytotoxicity kit. Cell phenotypic changes are analyzed through Western blot assays. Autophagy flux is monitored through the tandem red fluorescent protein–Green fluorescent protein–microtubule associated protein 1 light chain 3 (RFP–GFP–LC3) construct. The expression levels of DRAM1 and microtubule associated protein 1 light chain 3II/I (LC3II/I) are strongly up-regulated in Neuro-2a cells after OGD/R treatment and peaked at the 12 h reperfusion time point. The autophagy-specific inhibitor 3-Methyladenine (3-MA) inhibits the expression of DRAM1 and LC3II/I and exacerbates OGD/R-induced cell injury. Furthermore, DRAM1 knockdown aggravates OGD/R-induced cell injury and significantly blocks autophagy through decreasing autophagosome-lysosome fusion. In conclusion, our data demonstrate that DRAM1 knockdown in Neuro-2a cells inhibits autophagy by blocking autophagosome-lysosome fusion and exacerbated OGD/R-induced cell injury. Thus, DRAM1 might constitute a new therapeutic target for I/R diseases. View Full-Text
Keywords: DNA damage-regulated autophagy modulator protein 1 (DRAM1); oxygen-glucose deprivation and reperfusion (OGD/R); cerebral ischemia and reperfusion (I/R) injury; autophagy DNA damage-regulated autophagy modulator protein 1 (DRAM1); oxygen-glucose deprivation and reperfusion (OGD/R); cerebral ischemia and reperfusion (I/R) injury; autophagy
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Yu, M.; Jiang, Y.; Feng, Q.; Ouyang, Y.; Gan, J. DRAM1 Protects Neuroblastoma Cells from Oxygen-Glucose Deprivation/Reperfusion-Induced Injury via Autophagy. Int. J. Mol. Sci. 2014, 15, 19253-19264.

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