Acute NMDA toxicity in cultured rat cerebellar granule neurons is accompanied by autophagy induction and late onset autophagic cell death phenotype

Abstract

BackgroundAutophagy, 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).ResultsNMDA 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.ConclusionsCollectively, 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.

Effect of neuroserpin in a neonatal hypoxic-ischemic injury model ex vivo

“…However, it is important to indicate that the exact extent of excitotoxicity during neonatal HI is still undefi ned, and we are undecided whether the acute excitatory model used in our studies refl ects that seen in vivo. Previous research proves that acute NMDA toxicity is accompanied by autophagy induction and subsequent neuronal death (Sadasivan et al, 2010). In conjunction with this observation, our experiment also initiated a decrease of cell viability, a loss of neurons and an alteration of the neuronal morphology, which are detected by MAP2 expression.…”

Involvement of autophagy in hypoxic-excitotoxic neuronal death

“…Increased numbers of autophagosomes have been described in different in vitro situations of excitotoxicity such as long-term treatment and/or high doses of NMDA 30,31 or Ka, 19 or exposure to a specific glutamate uptake inhibitor. 32 The injection of toxic doses of Ka into the adult mice hippocampus 20,33 or striatum 34,35 also induces autophagy.…”

“…13,14,[36][37][38][39][40][41][42] Controversies remain, however, concerning the role of the autophagic activation in excitotoxic conditions both in vitro and in vivo. Even though most of the papers demonstrate a prodeath function of autophagy, [11][12][13][14]27,31,33,34,37,43,44 some studies suggest the opposite. 9,10,[45][46][47][48][49] These discrepancies could be due to differences in the excitotoxic conditions, to the use of pharmacological inhibitors with limited specificity 50,51 or to the lack of complete analysis on autophagy flux, i.e., demonstration that autophagosome increase is not due to lysosomal dysfunction in excitotoxic situations.…”

Neuronal Stimulation Induces Autophagy in Hippocampal Neurons That Is Involved in AMPA Receptor Degradation after Chemical Long-Term Depression

“…High doses and/or long incubation of NMDA are excitotoxic to neurons, and autophagy may be induced under these conditions in response to excitotoxicity (Borsello et al, 2003;Shacka et al, 2007;Chakrabarti et al, 2009;Sadasivan et al, 2010). However, the chem-LTD protocol was previously reported not to be excitotoxic (Li et al, 2010).…”