AMBRA1-Mediated Mitophagy Counteracts Oxidative Stress and Apoptosis Induced by Neurotoxicity in Human Neuroblastoma SH-SY5Y Cells.

Anthea Di Rita,Pasquale D’Acunzo,Luca Simula,Silvia Campello,Flavie Strappazzon,Francesco Cecconi

doi:10.3389/fncel.2018.00092

Abstract

Therapeutic strategies are needed to protect dopaminergic neurons in Parkinson’s disease (PD) patients. Oxidative stress caused by dopamine may play an important role in PD pathogenesis. Selective autophagy of mitochondria (mitophagy), mainly regulated by PINK1 and PARKIN, plays an important role in the maintenance of cell homeostasis. Mutations in those genes cause accumulation of damaged mitochondria, leading to nigral degeneration and early-onset PD. AMBRA1ActA is a fusion protein specifically expressed at the mitochondria, and whose expression has been shown to induce a powerful mitophagy in mammalian cells. Most importantly, the pro-autophagy factor AMBRA1 is sufficient to restore mitophagy in fibroblasts of PD patients carrying PINK1 and PARKIN mutations. In this study, we investigated the potential neuroprotective effect of AMBRA1-induced mitophagy against 6-hydroxydopamine (6-OHDA)- and rotenone-induced cell death in human neuroblastoma SH-SY5Y cells. We demonstrated that AMBRA1ActA overexpression was sufficient to induce mitochondrial clearance in SH-SY5Y cells. We found that apoptosis induced by 6-OHDA and rotenone was reversed by AMBRA1-induced mitophagy. Finally, transfection of SH-SY5Y cells with a vector encoding AMBRA1ActA significantly reduced 6-OHDA and rotenone-induced generation of reactive oxygen species (ROS). Altogether, our results indicate that AMBRA1ActA is able to induce mitophagy in SH-SY5Y cells in order to suppress oxidative stress and apoptosis induced by both 6-OHDA and rotenone. These results strongly suggest that AMBRA1 may have promising neuroprotective properties with an important role in limiting ROS-induced dopaminergic cell death, and the utmost potential to prevent PD or other neurodegenerative diseases associated with mitochondrial oxidative stress.

Highlights

Parkinson’s disease (PD) is a chronic and severe neurodegenerative disorder characterized by a progressive and selective death of dopaminergic neurons in the substantia nigra
We found that AMBRA1ActA surrounds aggregated mitochondria
In order to ascertain that mito-aggresome structures were specific for AMBRA1 mitochondrial localization coupled with a reduction of mitochondrial marker levels, we performed a confocal microscope analysis, in which SH-SY5Y cells were transfected with PcDNA3, Myc-AMBRA1ActA, VenusActA, a plasmid encoding the Actin assemblyinducing protein (ActA) sequence fused with the fluorescent reporter protein Venus (Nagai et al, 2002), and with the wild-type form of AMBRA1 lacking the ActA sequence

Summary

Introduction

Parkinson’s disease (PD) is a chronic and severe neurodegenerative disorder characterized by a progressive and selective death of dopaminergic neurons in the substantia nigra. Mitophagy is a self-degradative process that allows the elimination of ubiquitin-targeted mitochondria through lysosomal digestion Both PINK1 and PARKIN are key factors in mitophagy induction: PINK1 is a Ser-Thr kinase that mediates the phospho-ubiquitin signal, recruiting the E3 ubiquitin ligase PARKIN at the mitochondria. In the PD context, oxidative stress is widely considered to be a key factor in both familial and sporadic forms of the disease (Sanders et al, 2014). It results from an imbalance of pro-oxidants/antioxidants homeostasis that leads to an abnormal production of reactive oxygen species (ROS), whose overproduction generates damage of both neurons and astrocytes (Lin and Beal, 2006)

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Results

Conclusion