Acute NMDA toxicity in cultured rat cerebellar granule neurons is accompanied by autophagy induction and late onset autophagic cell death phenotype.
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.
Sadasivan
Shankar
S
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
Zhang
Zhiqun
Z
Larner
Stephen F
SF
Liu
Ming C
MC
Zheng
Wenrong
W
Kobeissy
Firas H
FH
Hayes
Ronald L
RL
Wang
Kevin K W
KK
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
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