title: Acute NMDA toxicity in cultured rat cerebellar granule neurons is accompanied by autophagy induction and late onset autophagic cell death phenotype.
abstract: BACKGROUND
  Autophagy, an intracellular response to stress, is characterized by double membrane cytosolic vesicles called autophagosomes. Prolonged autophagy is known to result in autophagic (Type II) cell death. This study examined the potential role of an autophagic response in cultured cerebellar granule neurons challenged with excitotoxin N-methyl-D-aspartate (NMDA).
  
  
  RESULTS
  NMDA exposure induced light chain-3 (LC-3)-immunopositive and monodansylcadaverine (MDC) fluorescent dye-labeled autophagosome formation in both cell bodies and neurites as early as 3 hours post-treatment. Elevated levels of Beclin-1 and the autophagosome-targeting LC3-II were also observed following NMDA exposure. Prolonged exposure of the cultures to NMDA (8-24 h) generated MDC-, LC3-positive autophagosomal bodies, concomitant with the neurodegenerative phase of NMDA challenge. Lysosomal inhibition studies also suggest that NMDA-treatment diverted the autophagosome-associated LC3-II from the normal lysosomal degradation pathway. Autophagy inhibitor 3-methyladenine significantly reduced NMDA-induced LC3-II/LC3-I ratio increase, accumulation of autophagosomes, and suppressed NMDA-mediated neuronal death. ATG7 siRNA studies also showed neuroprotective effects following NMDA treatment.
  
  
  CONCLUSIONS
  Collectively, this study shows that autophagy machinery is robustly induced in cultured neurons subjected to prolonged exposure to excitotoxin, while autophagosome clearance by lysosomal pathway might be impaired. Our data further show that prolonged autophagy contributes to cell death in NMDA-mediated excitotoxicity.
authors.affiliation: Center for Neuroproteomics and Biomarkers Research, Department of Psychiatry, McKnight Brain Institute of the University of Florida, Gainesville, FL 32610, USA. shankar.sadasivan@stjude.org
authors.forename: Shankar
authors.initials: S
authors.lastname: Sadasivan
authors.forename: Zhiqun
authors.initials: Z
authors.lastname: Zhang
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Larner
authors.forename: Ming C
authors.initials: MC
authors.lastname: Liu
authors.forename: Wenrong
authors.initials: W
authors.lastname: Zheng
authors.forename: Firas H
authors.initials: FH
authors.lastname: Kobeissy
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Hayes
authors.forename: Kevin K W
authors.initials: KK
authors.lastname: Wang
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects* / Q000502:physiology; D000071186:Beclin-1; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002491:Central Nervous System Agents / Q000494:pharmacology; D002531:Cerebellum / Q000187:drug effects* / Q000503:physiopathology; D008247:Lysosomes / Q000502:physiology; D016202:N-Methylaspartate / Q000633:toxicity*; D009410:Nerve Degeneration / Q000139:chemically induced / Q000503:physiopathology; D009474:Neurons / Q000187:drug effects* / Q000502:physiology; D009498:Neurotoxins / Q000633:toxicity*; D010641:Phenotype; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013997:Time Factors
chemical_list: D051017:Apoptosis Regulatory Proteins; D000071186:Beclin-1; C512829:Becn1 protein, rat; D002491:Central Nervous System Agents; D009498:Neurotoxins; C025946:3-methyladenine; D016202:N-Methylaspartate; C505410:Casp3 protein, rat; D053148:Caspase 3; D000225:Adenine