Pharmacological Inhibition of O-GlcNAc Transferase Promotes mTOR-Dependent Autophagy in Rat Cortical Neurons.

Md Ataur Rahman,Hyewhon Rhim,Hongik Hwang,Yoonjeong Cho

doi:10.3390/brainsci10120958

Abstract

O-GlcNAc transferase (OGT) is a ubiquitous enzyme that regulates the addition of β-N-acetylglucosamine (O-GlcNAc) to serine and threonine residues of target proteins. Autophagy is a cellular process of self-digestion, in which cytoplasmic resources, such as aggregate proteins, toxic compounds, damaged organelles, mitochondria, and lipid molecules, are degraded and recycled. Here, we examined how three different OGT inhibitors, alloxan, BXZ2, and OSMI-1, modulate O-GlcNAcylation in rat cortical neurons, and their autophagic effects were determined by immunoblot and immunofluorescence assays. We found that the treatment of cortical neurons with an OGT inhibitor decreased O-GlcNAcylation levels and increased LC3-II expression. Interestingly, the pre-treatment with rapamycin, an mTOR inhibitor, further increased the expression levels of LC3-II induced by OGT inhibition, implicating the involvement of mTOR signaling in O-GlcNAcylation-dependent autophagy. In contrast, OGT inhibitor-mediated autophagy was significantly attenuated by 3-methyladenine (3-MA), a blocker of autophagosome formation. However, when pre-treated with chloroquine (CQ), a lysosomotropic agent and a late-stage autophagy inhibitor, OGT inhibitors significantly increased LC3-II levels along with LC3 puncta formation, indicating the stimulation of autophagic flux. Lastly, we found that OGT inhibitors significantly decreased the levels of the autophagy substrate p62/SQSTM1 while increasing the expression of lysosome-associated membrane protein 1 (LAMP1). Together, our study reveals that the modulation of O-GlcNAcylation by OGT inhibition regulates mTOR-dependent autophagy in rat cortical neurons.

Highlights

The cycling of O-GlcNAcylation post-translational modification on the hydroxyl group of serine or threonine residues, known as O-GlcNAcylation, is catalyzed by O-GlcNAc transferase (OGT) andO-GlcNAcase (OGA) [1,2,3], which regulates essential cellular processes, including metabolism, transcription, protein–protein interaction, proteostasis, stress response, protein stability, subcellular localization, and autophagy [4,5,6,7]
To elucidate how O-GlcNAcylation modification affects autophagy in cortical neurons, we investigated the role of different OGT inhibitors in regulating the autophagic pathway
Cortical neurons were treated with an OGT inhibitor, and changes in O-GlcNAcylation levels and autophagic activities were examined by immunoblot and immunocytochemistry assays

Summary

Introduction

The cycling of O-GlcNAcylation post-translational modification on the hydroxyl group of serine or threonine residues, known as O-GlcNAcylation, is catalyzed by O-GlcNAc transferase (OGT) andO-GlcNAcase (OGA) [1,2,3], which regulates essential cellular processes, including metabolism, transcription, protein–protein interaction, proteostasis, stress response, protein stability, subcellular localization, and autophagy [4,5,6,7]. Brain Sci. 2020, 10, 958 it has been suggested that O-GlcNAcylation modification, mediated by OGT, exerts a neuroprotective function in neurodegenerative diseases by interfering with the production of toxic aggregates formed with α-synuclein, tau, amyloid precursor protein (APP) or neurofilament M (NFM) [9,10,11], and the cycling of O-GlcNAcylation was shown to modulate neurodegenerative phenotypes in Caenorhabditis elegans, such as amyloid β-peptide, polyglutamine, and tauopathy expansion. It remains to be examined whether O-GlcNAcylation directly contributes to the neuroprotective function by inducing autophagic activity in neurons, and the mechanisms by which O-GlcNAcylation regulates autophagic activity remain unknown at the molecular level. Revealing the association between autophagy and the regulation of O-GlcNAcylation in neuronal cells may provide essential implications for the treatment of neurodegenerative diseases

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