Multi-Omics after O-GlcNAc Alteration Identifies Cellular Processes Working Synergistically to Promote Aneuploidy.

Pharmacologic or genetic manipulation of O-GlcNAcylation, an intracellular, single sugar post-translational modification, are difficult to interpret due to the pleotropic nature of O-GlcNAc and the vast signaling pathways it regulates. To address this issue, we employed either OGT (O-GlcNAc transferase), OGA (O-GlcNAcase) liver knockouts, or pharmacological inhibition of OGA coupled with multi-Omics analysis and bioinformatics. We identified numerous genes, proteins, phospho-proteins, or metabolites that were either inversely or equivalently changed between conditions. Moreover, we identified pathways in OGT knockout samples associated with increased aneuploidy. To test and validate these pathways, we induced liver growth in OGT knockouts by partial hepatectomy. OGT knockout livers showed a robust aneuploidy phenotype with disruptions in mitosis, nutrient sensing, protein metabolism/amino acid metabolism, stress response, and HIPPO signaling demonstrating how OGT is essential in controlling aneuploidy pathways. Moreover, these data show how a multi-Omics platform can discern how OGT can synergistically fine-tune multiple cellular pathways.

O-GlcNAcylation regulates extracellular signal-regulated kinase (ERK) activation in Alzheimer's disease.

Introduction: Aberrant activation of Extracellular Signal-Regulated Kinase (ERK) signaling is associated with Alzheimer's disease (AD) pathogenesis. For example, enhanced ERK signal activation mediated by Apolipoprotein E4 (APOE4), which is a critical genetic risk factor for AD, increases the transcription of amyloid precursor protein (APP). We hypothesize that O-linked N-acetylglucosamine (O-GlcNAc) regulates the phosphorylation and activation of ERK. O-GlcNAc is a single sugar post-translational modification that dynamically cycles on and off proteins in response to nutrient changes by the action of the enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. However, O-GlcNAc quickly returns to a baseline level after stimulus removal (called O-GlcNAc homeostasis). Methods: We did a serum reactivation time-course followed by western blot in SH-SY5Y neuroblastoma cells after long-term O-GlcNAcase (OGA) inhibition by Thiamet-G (TMG) treatment, O-GlcNAc transferase (OGT) knock-down (KD) and OGA KD. Brain tissues of C57BL6/J mice and 5XFAD Alzheimer's disease mice intra-peritoneally injected with TMG for 1 month and C57BL6/J mice intra-peritoneally injected with TMG for 6 months were also used for western blot. Results: We found that ERK1/2 phosphorylation at Thr 202/Tyr204 and Thr183/Tyr185 (p-ERK) are amplified and hence ERK1/2 are activated after long-term OGA inhibition in SH-SY5Y cells. In addition to pharmacological treatment, genetic disruption of O-GlcNAc by OGT KD and OGA KD also increased p-ERK in SH-SY5Y cells suggesting O-GlcNAc homeostasis controls ERK signaling. To determine how O-GlcNAc regulates p-ERK, we probed the expression of phosphorylated mitogen-activated protein kinase-kinase (p-MEK) which phosphorylates and activates ERK and Dual specificity phosphatase-4 (DUSP4) which dephosphorylates and inactivates ERK in SH-SY5Y cells. p-MEK increases in TMG treated and OGT KD cells whereas total DUSP4 decreases in OGT KD and OGA KD cells with serum reactivation time course. Next, we probed the role of OGA inhibition in regulating ERK activation using mice brain-tissue samples. Interestingly, 6-month intra-peritoneal TMG injection in C57BL/6J mice showed an increase in amplitude of p-ERK and APP protein levels, indicating long-term OGA inhibition potentially contributes to AD progression. Furthermore, 1-month TMG injection was sufficient to increase the amplitude of p-ERK in 5XFAD AD mice brains suggesting AD phenotype contributes to the acceleration of ERK activation mediated by OGA inhibition. Conclusion: Together, these results indicate that disruptions to O-GlcNAc homeostasis amplify ERK signal activation in AD.

O-GlcNAc regulates the mitochondrial integrated stress response by regulating ATF4.

Background: Accumulation of mitochondrial dysfunctional is a hallmark of age-related neurodegeneration including Alzheimer's disease (AD). Impairment of mitochondrial quality control mechanisms leading to the accumulation of damaged mitochondria and increasing neuronal stress. Therefore, investigating the basic mechanisms of how mitochondrial homeostasis is regulated is essential. Herein, we investigate the role of O-GlcNAcylation, a single sugar post-translational modification, in controlling mitochondrial stress-induced transcription factor Activating Transcription Factor 4 (ATF4). Mitochondrial dysfunction triggers the integrated stress response (ISRmt), in which the phosphorylation of eukaryotic translation initiation factor 2α results in the translation of ATF4. Methods: We used patient-derived induced pluripotent stem cells, a transgenic mouse model of AD, SH-SY5Y neuroblastoma and HeLa cell-lines to examine the effect of sustained O-GlcNAcase inhibition by Thiamet-G (TMG) on ISRmt using biochemical analyses. Results: We show that TMG elevates ATF4 protein levels upon mitochondrial stress in SH-SY5Y neuroblastoma and HeLa cell-lines. An indirect downstream target of ATF4 mitochondrial chaperone glucose-regulated protein 75 (GRP75) is significantly elevated. Interestingly, knock-down of O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc, in SH-SY5Y increases ATF4 protein and mRNA expression. Additionally, ATF4 target gene Activating Transcription Factor 5 (ATF5) is significantly elevated at both the protein and mRNA level. Brains isolated from TMG treated mice show elevated levels of ATF4 and GRP75. Importantly, ATF4 occupancy increases at the ATF5 promoter site in brains isolated from TMG treated mice suggesting that O-GlcNAc is regulating ATF4 targeted gene expression. Interestingly, ATF4 and GRP75 are not induced in TMG treated familial Alzheimer's Disease mice model. The same results are seen in a human in vitro model of AD. Conclusion: Together, these results indicate that in healthy conditions, O-GlcNAc regulates the ISRmt through regulating ATF4, while manipulating O-GlcNAc in AD has no effect on ISRmt.

Mapping the O-GlcNAc Modified Proteome: Applications for Health and Disease.

O-GlcNAc is a pleotropic, enigmatic post-translational modification (PTM). This PTM modifies thousands of proteins differentially across tissue types and regulates diverse cellular signaling processes. O-GlcNAc is implicated in numerous diseases, and the advent of O-GlcNAc perturbation as a novel class of therapeutic underscores the importance of identifying and quantifying the O-GlcNAc modified proteome. Here, we review recent advances in mass spectrometry-based proteomics that will be critical in elucidating the role of this unique glycosylation system in health and disease.