Abstract
O-linked conjugation of ß-N-acetyl-glucosamine (O-GlcNAc) to serine and threonine residues is a post-translational modification process that senses nutrient availability and cellular stress and regulates diverse biological processes that are involved in neurodegenerative diseases and provide potential targets for therapeutics development. However, very little is known of the networks involved in the brain that are responsive to changes in the O-GlcNAc proteome. Pharmacological increase of protein O-GlcNAcylation by Thiamet G (TG) has been shown to decrease tau phosphorylation and neurotoxicity, and proposed as a therapy in Alzheimer’s disease (AD). However, acute TG exposure impairs learning and memory, and protein O-GlcNAcylation is increased in the aging rat brain and in Parkinson’s disease (PD) brains. To define the cortical O-GlcNAc proteome that responds to TG, we injected young adult mice with either saline or TG and performed mass spectrometry analysis for detection of O-GlcNAcylated peptides. This approach identified 506 unique peptides corresponding to 278 proteins that are O-GlcNAcylated. Of the 506 unique peptides, 85 peptides are elevated by > 1.5 fold in O-GlcNAcylation levels in response to TG. Using pathway analyses, we found TG-dependent enrichment of O-GlcNAcylated synaptic proteins, trafficking, Notch/Wnt signaling, HDAC signaling, and circadian clock proteins. Significant changes in the O-GlcNAcylation of DNAJC6/AUXI, and PICALM, proteins that are risk factors for PD and/or AD respectively, were detected. We compared our study with two key prior O-GlcNAc proteome studies using mouse cerebral tissue and human AD brains. Among those identified to be increased by TG, 15 are also identified to be increased in human AD brains compared to control, including those involved in cytoskeleton, autophagy, chromatin organization and mitochondrial dysfunction. These studies provide insights regarding neurodegenerative diseases therapeutic targets.
Highlights
In this study we first assessed the impact on total levels of protein O-GlcNAcylation in response to TG using the lower resolution western blotting techniques and found a robust 3-fold increase in protein O-GlcNAcylation (Figure 1)
Using the isobaric tandem mass tag labelling combined with chemoenzymatic photocleavage (CEPC) method we examined the O-GlcNAc proteome with and without the i. p administration of the inhibitor of OGA TG (Wang et al., 2017)
We identified 506 O-GlcNAcylated peptides, and of these, 85 peptides corresponding to 65 proteins were at least 50% more O-GlcNAcylated from the TG treated mouse cortex compared to saline control
Summary
First discovered in the 1980s, the O-GlcNAcylation of proteins is widely accepted as playing a key role in the regulation of diverse biological processes integrating nutrient availability and cellular stress (Zachara and Hart, 2004; Love and Hanover, 2005; Slawson et al, 2006; Paruchuri and Zachara, 2011; Darley-Usmar et al, 2012; Bond and Hanover, 2013; Alonso et al, 2014; Hardivillé and Hart, 2014; Marsh et al, 2014; Wani et al, 2015). Pharmacological approaches to increase O-GlcNAc levels, such as inhibition of O-GlcNAcase (OGA) by Thiamet G (TG), increased tau O-GlcNAcylation and decreased tau phosphorylation and associated neurodegenerative phenotypes (Yuzwa et al, 2008; Yuzwa et al, 2012; Yuzwa et al, 2014; Zhu et al, 2014; Hastings et al, 2017). O-GlcNAc modification of 85 peptides corresponding to 65 proteins increased at >1.5-fold in the TG treatment group compared to controls Network analysis of these changes revealed that pathways involved in synaptic function are enriched. These findings suggest that DNAJC6 and PICALM O-GlcNAcylation are potential contributing factors to neurodegenerative diseases
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