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  • Modulation of O-GlcNAcylation Regulates Autophagy in Cortical Astrocytes.

Modulation of O-GlcNAcylation Regulates Autophagy in Cortical Astrocytes.

Oxidative medicine and cellular longevity (2019-12-13)
Md Ataur Rahman, Hongik Hwang, Yoonjeong Cho, Hyewhon Rhim
ABSTRACT

The addition of O-linked β-N-acetylglucosamine (O-GlcNAcylation) to serine and threonine residues is a common posttranslational modification of intracellular proteins which modulates protein functions and neurodegenerative diseases, controlled by a single pair of enzymes, O-GlcNAcase (OGA), and O-GlcNAcylation transferase (OGT). Autophagy is a cellular recycling pathway activated by stress and nutrient signaling; however, the mechanism by which O-GlcNAcylation modification regulates autophagy in cortical astrocytes is poorly understood. Here, we report that increased O-GlcNAcylation by the suppression of OGA activity using thiamet-G and OGA siRNA did not affect autophagy, whereas decreased O-GlcNAcylation caused by OGT inhibition by alloxan and OGT siRNA increased autophagy. OGT inhibitor and siRNA accumulated LC3 puncta, and cotreatment with chloroquine (CQ), an autophagy inhibitor, significantly increased LC3 puncta and LC3-II protein, confirming that decreased O-GlcNAcylation promotes autophagic flux. In particular, we found that OGT knockdown increases the fusion between autophagosomes as well as lysosomes and stimulates autophagy to promote lysosomal-associated membrane protein 1 (LAMP-1). Additionally, decreasing O-GlcNAcylation by treatment with alloxan, OGT siRNA, and OGA overexpression significantly decreased the level of autophagy substrate SQSTM1/p62, indicating that autophagic degradation was activated. Together, our study reveals a mechanism by which the modulation of O-GlcNAcylation modification regulates autophagy in mouse cortical astrocytes.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
MISSION® esiRNA, targeting human MGEA5
Sigma-Aldrich
MISSION® esiRNA, targeting human OGT
Sigma-Aldrich
Chloroquine diphosphate salt, powder or crystals, 98.5-101.0% (EP)