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.
Highlights
O-linked-N-acetylglucosamine (O-GlcNAcylation), called O-GlcNAcylation, is an abundant posttranslational modification of multiple proteins proposed to have both neurodegenerative as well as neuroprotective functions [1]
To understand the process of autophagy, it is important to know the molecular role of microtubule-associated protein 1 light chain 3 (LC3) which is a ubiquitin-like modifier protein generally involved in autophagosome biogenesis in autophagy signaling
Immunofluorescence staining with an anti-LC3 antibody showed that the number of LC3 puncta per cells was not changed by thiamet-G even dose-dependently, whereas alloxan-treated astrocytes significantly increased LC3 puncta (Figures 1(c) and 1(d))
Summary
O-linked-N-acetylglucosamine (O-GlcNAcylation), called O-GlcNAcylation, is an abundant posttranslational modification of multiple proteins proposed to have both neurodegenerative as well as neuroprotective functions [1]. Previous studies indicate that O-GlcNAcylation regulates essential processes, such as metabolism [2], stress response [3], transcription [4], proteostasis [5], and autophagy [6]. A study of Caenorhabditis elegans suggests that increasing O-GlcNAcylation inhibits autophagy [6], whereas decreasing O-GlcNAcylation activates autophagic flux by mutant huntingtin protein [8]. It has been found that OGA inhibitors prompted autophagy in two mouse models of Alzheimer’s disease (AD) as well as in primary neuron culture [10]. Increasing O-GlcNAcylation levels by pharmacological inhibition of OGA decreases β-amyloid deposition, motor deficits, phosphorylation of tau, and impairments of memory function in certain mouse models of AD [11]. Despite the prevalence of O-GlcNAcylation modification, Oxidative Medicine and Cellular Longevity understanding the molecular basis of O-GlcNAcylationregulated autophagic activity still requires further investigation
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