title: DRAM1 protects neuroblastoma cells from oxygen-glucose deprivation/reperfusion-induced injury via autophagy.
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.
authors.affiliation: Department of Neurosurgery, Second Xiangya Hospital of Central South University, Changsha 410011, China. yumengqiang@live.cn.
authors.forename: Mengqiang
authors.initials: M
authors.lastname: Yu
authors.affiliation: Department of Neurosurgery, Second Xiangya Hospital of Central South University, Changsha 410011, China. jyg028@163.com.
authors.forename: Yugang
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Department of Neurosurgery, Second Xiangya Hospital of Central South University, Changsha 410011, China. fengqingliang028@163.com.
authors.forename: Qingliang
authors.initials: Q
authors.lastname: Feng
authors.affiliation: Department of Neurosurgery, Second Xiangya Hospital of Central South University, Changsha 410011, China. ouyangyian028@163.com.
authors.forename: Yi'an
authors.initials: Y
authors.lastname: Ouyang
authors.affiliation: Department of Neurosurgery, Second Xiangya Hospital of Central South University, Changsha 410011, China. ganjie028@163.com.
authors.forename: Jie
authors.initials: J
authors.lastname: Gan
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D002545:Brain Ischemia / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D005947:Glucose / Q000378:metabolism*; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D015427:Reperfusion Injury / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D051017:Apoptosis Regulatory Proteins; C572524:DRAM-1 protein, mouse; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; C025946:3-methyladenine; D005947:Glucose; D000225:Adenine; D010100:Oxygen