O-GlcNAcylation of serine or threonine residues is a dynamic, inducible PTM of intracellular proteins that
regulates multiple physiological functions including insulin signaling,1 transcription,2 mitosis,3 metabolism,4 and neuronal homeostasis.5 Unlike other forms of N- and O-glycosylation, which are often made up of extensive, intricately branching polymers, O-GlcNAcylation is never elaborated beyond the single GlcNAc monomer. To date, O-GlcNAcylation has been observed in all metazoans and over 1,000 O-GlcNAcylated proteins have been described in higher mammals and plants.6,7
Despite this incredible substrate diversity, there are only two enzymes known to be responsible for addition and removal of O-GlcNAc in mammals, OGT and OGA, respectively (Fig. 1.1).6 Notably, OGT knockout (KO) is embryonically lethal in mice and is necessary for survival in dividing mammalian cells.8,9 OGA KO is also perinatal lethal, although in rare cases mice survive to adulthood with profound metabolic abnormalities.10-12 Given the widespread, critical, and specific regulatory nature of this modification, the fact that only two enzymes are responsible for its cycling suggests exquisite mechanisms for controlling the activity and targeting of these enzymes. Accordingly, dysregulation of O-GlcNAc cycling has been linked to numerous human diseases such as diabetes,13 cancer,14 and neurodegeneration.15
The OGT gene is located on the X-chromosome (Xq13.1 in humans) and is expressed as three distinct isoforms generated by alternative splicing: nucleocytoplasmic (ncOGT), mitochondrial (mOGT), and short (sOGT).8,168,9 The three OGT isoforms are each composed of a conserved C-terminal catalytic domain and differ by the number of N-terminal tetratricopeptide
Fig. 1.1. O-GlcNAc Cycling by OGT and OGA
O-GlcNAcase (OGA)
O-GlcNAc Transferase (OGT)
(TPR) repeats. OGA is encoded by MGEA5 on chromosome 10 (10q23.1-23.4 in humans) and has two isoforms: full-length OGA (fOGA) and short (sOGA), which are predominantly localized to the cytosol and nucleus, respectively.17-19 fOGA and sOGA differ by the presence or absence of a C-terminal histone acetyltransferase domain.20 Crystal structures of both human OGT 21,22 and OGA 23-25 have been recently described, and insights into structure-function relationships have been reviewed elsewhere.6,20,26-28
Understanding the specifics of O-GlcNAc cycling on different substrates is key to understanding the overall functions of O-GlcNAc on a cellular and organismal scale. Moreover, it may lead to novel therapeutic interventions in human disease.13,15 In higher mammals, O- GlcNAcylation and its cycling enzymes are most highly expressed in the pancreas and brain.6,15 OGT activity was also shown to be 10-fold higher in the brain compared to other tissues,29 and there have been thousands of O-GlcNAc sites identified on proteins intimately involved with neuronal function.30-35 Importantly, there is also a strong association between aberrant O- GlcNAcylation and AD and other neurodegenerative diseases.15 Thus, given the immense costs and rapidly increasing prevalence of AD, along with absence of disease-modifying treatments, understanding the function and regulation of O-GlcNAcylation in the brain is perhaps one of the most pressing issues in modern biomedical research.36,37
Within the brain, OGT and OGA expression exhibits temporal and spatial variability across substructures, with the highest levels occurring within the cortex, hippocampus, and cerebellum.29,38-40 At the cellular level, OGT and OGA, along with O-GlcNAc, are highly enriched in the nuclear and synaptosomal fractions of neurons.39,41,42 Interestingly, while O-GlcNAcylation and OGT are also enriched in the postsynaptic density (PSD), OGA is excluded.43 Furthermore, O-GlcNAc and OGT/OGA expression have been observed in multiple types of excitatory and
inhibitory neurons as well as astrocytes, oligodendrocytes, and microglia.40,44 Overall, the aforementioned observations have led to intense investigation of the role of O-GlcNAc in neuronal function, the results of which have recently been well-summarized previously.12,45
In some of the same studies, reporting aberrant O-GlcNAcylation in AD, alterations in O- GlcNAcylation were also found at earlier time points, including in individuals with mild cognitive impairment and T2DM.46,47 O-GlcNAcylation has also been found to be elevated across multiple organ systems in T2DM, including peripheral fat tissue, muscle, liver, pancreas, and kidneys.48-50 It has long been hypothesized that O-GlcNAc serves as a sensor for extracellular glucose concentrations and can direct intracellular response accordingly.51 Importantly, elevated O- GlcNAcylation in response to hyperglycemia has been mechanistically linked to kidney, cardiac, and pancreatic complications of T2DM.46,52-54 In the liver, increased O-GlcNAcylation has also been demonstrated to play a major role in maintaining organismal glucose homeostasis.50 Moreover, T2DM has a strong epidemiological link with AD and other dementias,55-58 and the changes in O-GlcNAcylation and associated dysfunction across organ systems seen with T2DM may potentially set the stage for the development of brain pathology.15 Altogether, it is becoming increasingly clear that O-GlcNAcylation is intimately associated with cellular and organismal homeostasis and is likely a major player in the pathophysiology of metabolic and neurodegenerative diseases.
In spite of the wealth of information that has already been gathered on O-GlcNAcylation, many studies up to this point have almost exclusively taken a reductionist approach, trying to understand the role of O-GlcNAcylation on the function of a single protein2 or exploring how modulating global O-GlcNAc levels affects a limited number of selected proteins or flux through a single signaling pathway.42 However, unravelling exactly how O-GlcNAc cycling enzymes
recognize, discriminate, and act upon specific protein substrates in response to specific stimuli is critical to our understanding of the functions of O-GlcNAcylation in neurons, a task which has been largely ignored. For example, OGT has been shown to preferentially glycosylate neurofilament proteins in response to glucose deprivation; a preference arising from an increased affinity to p38 mitogen-activated protein kinase (MAPK).59 Moreover, the observation that OGT interacts and participates in a functional complex with trafficking kinesin proteins (TRAKs) 1/260,61 led to the discovery that O-GlcNAcylation regulates mitochondrial motility in response to glucose availability in neurons.62 In fact, it has been proposed that the interactions of OGT/OGA with various binding proteins, scaffolds, and lipids allow them to act specifically on coordinated groups of proteins to differentially respond to stimuli, known as the adaptor protein hypothesis.27,63 Taken with the multitude of examples of a regulatory role for O-GlcNAcylation/OGT in multiprotein complexes, well-outlined in a recent review,26 these data suggest that identifying and characterizing the dynamics of OGT/OGA interacting partners may provide critical insight into the functions of O-GlcNAc in neurons.
Herein, we will review the previous literature on the role of O-GlcNAcylation in metabolism and neuronal function, with a particular focus on the role of O-GlcNAcylation in maintaining glucose homeostasis in the liver and its role in neuronal signaling and neuroprotection.
In the liver, we will outline the role of O-GlcNAcylation in insulin resistance, gluconeogenesis, and lipid metabolism. In neurons, we will discuss modulation of neuronal signaling, synaptic plasticity and cognition, neuroprotection, and neurodegenerative diseases as potential overarching functions of O-GlcNAc. Overall, the continued elucidation of the functional roles of O-GlcNAc in physiology and disease will undoubtedly lead to a deeper understanding across multiple fields of biology.
1.3. The Role of O-GlcNAc in Metabolic