Objective: In this study, we investigated the role of autophagy system in MANF-mediated neuroprotection against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity.

Background: Autophagy and apoptosis are commonly involved in the dopaminergic neuron damage in the pathogenesis of Parkinson’s disease. Recently, the autophagy pathway is thought to be important in the process of PD, and the regulation of autophagy may be a potential strategy for PD treatment. Mesencephalic astrocyte-derived neurotrophic factor (MANF) has been reported to have neuroprotective effects through anti-apoptosis, anti-oxidative, and anti-inflammatory mechanisms in PD. However, whether autophagic regulation is involved in MANF-mediated neuroprotection remains need to be elucidated.

Methods: The viability and apoptosis of SH-SY5Y cells were assessed by 3-(4, 5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay and Annexin V-FITC and propidium iodide (PI) double staining. Generation of intracellular reactive oxygen species (ROS) and superoxide anion were visualized and analyzed using oxidation sensitive probe DCFH-DA and DHE probes, respectively. A fluorescent dye JC-1 was applied to monitor mitochondrial membrane potential, while the intracellular ATP level was measured by ATP assay kit. The expression of autophagy related proteins were detected by western blot.

Results: MANF protected SH-SY5Y cells against 6-OHDA induced cell viability decrease and apoptosis by inhibiting autophagy. Reactive oxidative stress (ROS) accumulation triggered mitochondrion damage and energetic dysfunction were also alleviated by MANF treatment. Meanwhile, MANF downregulated phosphorylation of AMP-activated protein kinase (AMPK), a cellular energy sensor and regulator, but upregulated phosphorylation of Mammalian target of rapamycin (mTOR) under energy depletion conditions, indicating AMPK/mTOR signaling pathway was involved in the autophagic inhibition of MANF.

Conclusions: Our results suggest that autophagic inhibition is a protective mechanism of MANF in 6-OHDA induced SH-SY5Y cell death and this inhibition was associated with AMPK/mTOR pathway.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/mesencephalic-astrocyte-derived-neurotrophic-factor-alleviated-6-ohda-induced-cell-damage-via-ros-ampkmtor-mediated-autophagic-inhibition/