Autophagy activation is involved in 3,4-methylenedioxymethamphetamine ('ecstasy')--induced neurotoxicity in cultured cortical neurons.

I-Hsun Li,Shiang-Suo Huang,Chang-Min Liang,Kuo-Hsing Ma,Shao-Ju Weng,Yuahn-Sieh Huang,Udai Pandey

doi:10.1371/journal.pone.0116565

I-Hsun Li, Shiang-Suo Huang + Show 5 more

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https://doi.org/10.1371/journal.pone.0116565

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Abstract

Autophagic (type II) cell death, characterized by the massive accumulation of autophagic vacuoles in the cytoplasm of cells, has been suggested to play pathogenetic roles in cerebral ischemia, brain trauma, and neurodegenerative disorders. 3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is an illicit drug causing long-term neurotoxicity in the brain. Apoptotic (type I) and necrotic (type III) cell death have been implicated in MDMA-induced neurotoxicity, while the role of autophagy in MDMA-elicited neurotoxicity has not been investigated. The present study aimed to evaluate the occurrence and contribution of autophagy to neurotoxicity in cultured rat cortical neurons challenged with MDMA. Autophagy activation was monitored by expression of microtubule-associated protein 1 light chain 3 (LC3; an autophagic marker) using immunofluorescence and western blot analysis. Here, we demonstrate that MDMA exposure induced monodansylcadaverine (MDC)- and LC3B-densely stained autophagosome formation and increased conversion of LC3B-I to LC3B-II, coinciding with the neurodegenerative phase of MDMA challenge. Autophagy inhibitor 3-methyladenine (3-MA) pretreatment significantly attenuated MDMA-induced autophagosome accumulation, LC3B-II expression, and ameliorated MDMA-triggered neurite damage and neuronal death. In contrast, enhanced autophagy flux by rapamycin or impaired autophagosome clearance by bafilomycin A1 led to more autophagosome accumulation in neurons and aggravated neurite degeneration, indicating that excessive autophagosome accumulation contributes to MDMA-induced neurotoxicity. Furthermore, MDMA induced phosphorylation of AMP-activated protein kinase (AMPK) and its downstream unc-51-like kinase 1 (ULK1), suggesting the AMPK/ULK1 signaling pathway might be involved in MDMA-induced autophagy activation.

Highlights

Macroautophagy is a highly conserved cellular catabolic process whereby organelles and soluble and aggregated cellular components are enveloped in double-membrane vesicles called autophagosomes, which eventually fuse with lysosomes, leading to the degradation and reuse of the vesicular contents [1,2,3,4,5]
Cortical neuron cultures were treated with different concentration of MDMA (0.5, 1, 1.5, 2 mM) for 48 h, and the cell viability was determined by MTT assay
Treatment with 0.5 and 1 mM MDMA for 48 h did not have an obvious effect on the cell viability, as approximately 95% and 91% of the cell viability compared to normal control

Summary

Introduction

Macroautophagy ( referred to as autophagy) is a highly conserved cellular catabolic process whereby organelles and soluble and aggregated cellular components are enveloped in double-membrane vesicles called autophagosomes, which eventually fuse with lysosomes, leading to the degradation and reuse of the vesicular contents [1,2,3,4,5]. Autophagy occurs constitutively at a basal level in all eukaryotic cells and operates as a homeostatic mechanism [6]. In this role, autophagy removes unwanted cellular structures by the degradation of excess or damaged organelles and proteins and thereby contributes to the routine turnover of cytoplasmic components [7]. Defective autophagy has been connected to many human diseases including cancer, myopathies, and neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis [5, 11, 12]

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