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

Shankar Sadasivan,Ronald L Hayes,Ming C Liu,Kevin Kw Wang,Kevin Kw Wang,Kevin Kw Wang,Stephen F Larner,Stephen F Larner,Zhiqun Zhang,Zhiqun Zhang,Wenrong Zheng,Ming C Liu,Firas H Kobeissy,Ronald L Hayes

doi:10.1186/1471-2202-11-21

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.

Highlights

Autophagy, an intracellular response to stress, is characterized by double membrane cytosolic vesicles called autophagosomes
To assess the possible induction of autophagy following acute NMDA exposure, the neurons were stained with an antibody against microtubule associated light chain-3 (LC3) protein, a known autophagy protein marker, called Atg-8 [23]
Autophagy protein markers beclin-1 and LC3-II are upregulated following early phase of NMDA exposure Having established the induction of autophagy in neurons exposed to NMDA, we sought to study protein levels of the autophagy protein marker beclin-1 (Atg6)

Summary

Introduction

An intracellular response to stress, is characterized by double membrane cytosolic vesicles called autophagosomes. Autophagy is an intracellular pathway that is activated in response to cell stress It is a phenomenon where the cytoplasmic organelles in the cell are engulfed by double membrane vesicles called the autophagosomes and delivered to the lysosomes where the organelles are broken down by lysosomal proteases and the amino acids recycled back into the cell machinery to aid cell survival [1,2]. An elevated level of Bcl-2-binding protein Beclin-1 (Atg6) has been documented to be indicative of autophagy induction. Another protein marker for autophagy induction extensively studied, is the lipidated form of microtubule associated protein light chain-3 (MAP-LC3) found on the outer and to a lesser extent the inner membrane of the double membrane of the autophagosome

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Results

Conclusion