investigations into the role of O-GlcNAc in these and other disorders, including elucidating the full landscape of O-GlcNAcylated proteins and how it changes in response to disease state, are thus likely to reveal new avenues for therapeutic intervention.
Globally increasing O-GlcNAcylation has also been demonstrated to limit cognitive decline and neuronal pathology in mouse models of neurodegeneration.15,83,101-103 Overall, it is becoming increasingly clear that O-GlcNAcylation plays a pivotal role in neuronal health and function, and modulating O-GlcNAc signaling may represent a promising neuroprotective strategy for neurodegenerative diseases.12,15
1.5.1. Modulation of Neuronal Signaling.
Given that a major function of neurons is to integrate, respond to, and relay extracellular signals, it is perhaps not surprising that many studies have uncovered a functional role for O- GlcNAcylation in this area. The regulation of protein phosphorylation, the canonical mode of signal transduction in neurons,104-106 by O-GlcNAc is a common theme, especially in regards to interplay at the site and protein level.6,107,108 In fact, a major function of O-GlcNAcylation in neuronal signaling may simply be to oppose phosphorylation. For instance, Hart and coworkers have long advanced the idea that O-GlcNAcylation and phosphorylation exist in a yin-yang relationship,6,107-109 and there are numerous examples of direct yin-yang relationships between phosphorylation and O-GlcNAcylation in regulating the function, binding, and activity of individual proteins.99,110-115 Moreover, inhibition or activation of multiple kinases and phosphatases were shown to, in general, reciprocally modulate global O-GlcNAcylation (i.e.
treatments that generally increased global phosphorylation reduced, and treatments decreasing phosphorylation increased, O-GlcNAcylation) in cultured mouse cerebellar neurons, with the most significant effects seen in the cytoskeletal associated fraction.116 However, proteome-wide analyses of O-GlcNAcylation and phosphorylation have revealed a more complicated picture. In an analysis of proteins isolated from mouse cortical homogenate, only 24% of all mapped O-
GlcNAc sites were either within 10 amino acids of or themselves a known phosphorylation site.33 In another study which isolated both O-GlcNAcylated and phosphorylated proteins from murine synaptosomes, O-GlcNAcylation and phosphorylation sites were not correlated (outside of both being more common in disordered regions), and less than 7% of all O-GlcNAc sites were also phosphorylation sites.34 In an in vitro, peptide glycosylation assay, OGT also did not recognize the proline-directed kinase motif, PX(S/T)P, which makes up ~40% of all known phosphorylation sites.117 However, the same study described a well-defined “interplay motif,”
(pS/pT)P(V/A/T)(gS/gT), where O-GlcNAcylation is strongly inhibited by phosphorylation.
Overall, while the above data suggest that while direct competition for the same or close by serine/threonine residues is likely a minor phenomenon on the scale of the phosphoproteome, there is clearly a certain subset of O-GlcNAcylation/phosphorylation events that exist in a yin-yang relationship.
At the protein level, virtually all O-GlcNAcylated proteins are known to also be phosphorylated, and the subset of O-GlcNAcylated proteins are enriched for kinases as a class.33-
35 Globally increasing O-GlcNAcylation also increases the phosphorylation of synapsin extracellular signal-regulated kinase (ERK) 1/2 in hippocampal slices.42 Thus, O-GlcNAc may be playing a more active role in regulating functional aspects of neuronal kinases and signaling cascades. To date, many kinases heavily implicated in learning and memory, such as calcium- calmodulin dependent kinase (CaMK) II,46 CaMKIV,113 and PKA95 have been shown to be functionally regulated by O-GlcNAcylation. We have also found that O-GlcNAcylation and phosphorylation occur simultaneously in distinct locations on CREB, and interestingly, phosphorylated CREB is preferentially O-GlcNAcylated in response to neuronal activity.2 Taken together, these data suggest that O-GlcNAcylation may be playing a broader role in coordinating
the activity of multiple kinases and their substrates in ways other than directly opposing phosphorylation.
Next, we discuss examples of how O-GlcNAcylation regulates, and is regulated by, calcium signaling, a major subset of the neuronal signally machinery and absolutely required for long-term potentiation (LTP) and memory formation.118-120 In neurons, calcium levels are mediated by the flux of calcium into the cell primarily through voltage-operated channels (VOCs), and the release of calcium from intracellular stores mediated by receptor-operated channels (ROCs) and store-operated channels (SOCs). Major targets of calcium signaling include the CaMKs, a family of serine/threonine kinases expressed throughout the brain and are involved in neuronal excitability and plasticity.121 This class of kinases is activated by increases in the cytosolic concentration of calcium, resulting in phosphorylation of their substrates and activation of downstream signaling events. Increasing evidence suggests an intimate relationship between CaMK-mediated calcium signaling and O-GlcNAcylation.
In neuroblastoma cells, potassium chloride-induced depolarization across the membrane results in an increase in the activity of OGT, leading to an increase in O-GlcNAcylation of protein substrates.122 Interestingly, this effect could be prevented by inhibition of voltage-operated calcium channels, indicating that these effects on O-GlcNAcylation were mediated by calcium signaling pathways.122 Furthermore, inhibition of CaMKs also abolished the previously observed increase in O-GlcNAcylation indicating that CaMKs are involved in regulating the activity of OGT.122 Of the CaMKs, CaMKIV activity was found to upregulate phosphorylation of OGT upon depolarization of cells, resulting in an increase in OGT activity. While CaMKIV may regulate OGT activity, evidence indicating a role for OGT in regulating the activity of CaMKIV has also emerged. In cells, CaMKIV can be O-GlcNAcylated in the active site, an event which opposes its
phosphorylation and activation 113. Importantly, O-GlcNAcylation of CaMKIV also impeded activation of downstream signaling events such as activation of CREB, highlighting OGT as a regulator of CaMKIV-mediated calcium signaling pathways.
Similar interplay between calcium signaling and O-GlcNAcylation may also exist along a CaMKII-mediated axis. In 2013, it was demonstrated that diabetic hyperglycemia induced O- GlcNAcylation of CaMKII in the heart and brain, resulting in chronic activation of CaMKII- dependent pathways. In cardiomyocytes, these effects contributed to dysfunctional spontaneous release of calcium from intracellular stores, contributing to arrhythmias. Importantly, these effects were rescued by inhibition of O-GlcNAcylation, suggesting a regulatory role for O-GlcNAc in CaMKII-mediated calcium signaling.46 More recently, it was demonstrated that CaMKII phosphorylates OGT and that this modification has some influence on substrate-specificity of OGT.89 While performed in liver cells, this study highlights the role of calcium-signaling as a means through which O-GlcNAcylation levels are regulated and CaMKII-mediated activation of OGT may be important in excitable cells, similar to that observed of CaMKIV-mediated activation.
Together, the above studies highlight a delicate interplay which is present in calcium-mediated and OGT-mediated signaling pathways and suggest there may exist feed-back mechanisms;
however, future studies will be needed to probe CaMKII-OGT and CaMKIV-OGT regulatory axes in order to further explore these mechanisms.
A role for O-GlcNAcylation in regulating store-operated calcium signaling was highlighted by early studies demonstrating that increases in hexosamine biosynthesis pathway flux and increases in O-GlcNAc levels by treatment with extracellular glucosamine dampened store- operated calcium entry (SOCE) in excitable cells, such as cardiomyocytes.123 Furthermore, the metabolic state of the cell exerts dramatic effects on SOCE; for example, hyperglycemia was found
to dampen SOCE and that concomitant inhibition of the hexosamine biosynthesis pathway by azaserine (a potent inhibitor of GFAT) restored SOCE.123
Recent studies aimed at identifying mechanisms through which O-GlcNAcylation may be mediating these effects have focused on the ER-membrane protein and calcium sensor stromal interaction molecule 1 (STIM1). Upon depletion of calcium stores in the ER, STIM1 adopts an extended, active conformation.124-127 Active STIM1 oligomerizes and translocates to pre-existing endoplasmic reticulum (ER)-plasma membrane junctions,128,129 leading to recruitment of ORAI1 and activation of SOCE.127,130 These STIM1-ORAI1 complexes mediate calcium-selective influx of ions into the cell. Importantly, the O-GlcNAcylation of STIM1 was found to inhibit STIM1 mediated SOCE. O-GlcNAcylation may play a protective role in calcium signaling based on the findings that acute increases in O-GlcNAc levels prevent Ca overload induced by the calcium paradox characteristic of ischemia and reperfusion injuries 131.
1.5.2. Synaptic Plasticity and Cognition.
Having highlighted some examples of how O-GlcNAcylation may be involved in regulating neuronal signaling cascades, we next turn to the discussion of O-GlcNAc’s roles in higher-order neuronal functions. We start on the synaptic level with the emerging role of O- GlcNAcylation in regulating AMPAR trafficking. We then move to the role of O-GlcNAcylation in LTP and long-term depression (LTD) of excitatory synapses, beyond its role in regulating AMPAR trafficking, the likely cellular correlates of memory formation and maintenance. Finally, we review the first evidence that site specific O-GlcNAcylation can influence memory formation and recall in vivo and discuss the other examples of how changes in global O-GlcNAcylation can affect behavior.
AMPAR are ligand-gated (glutamate) cation channels responsible for the majority of excitatory neurotransmission in the mammalian brain.132 Moreover, they are intimately connected to learning and memory as their dynamic insertion into and removal from the postsynaptic membrane (trafficking) is thought to be the major means by which neurons potentiate or depress individual synapses.132,133 Thus, the interesting observation by Vosseller and coworkers that inhibiting OGA/OGT enhanced/inhibited LTP (discussed further below),42 strongly suggested that global modulation of O-GlcNAc levels likely has a strong effect on AMPAR trafficking. Shortly after the aforementioned observation, Din et al. hypothesized that O-GlcNAc may oppose known, functionally relevant phosphorylation events on AMPAR subunits GluA1 and GluA2 to maintain LTD and LTP respectively.134 Thereafter, Kanno et al. reported that treatment with the OGT inhibitor alloxan resulted in the increased insertion of both GluA1 and GluA2 into the plasma membrane.135 Another study reported that GluA2, but not GluA1, was O-GlcNAc modified, and that this modification was independent of S880 phosphorylation by PKC, a well-established mediator of AMPAR internalization.40 Finally, culturing embryonic cortical neurons harvested from floxed-OGT mice9 allowed Huganir and colleagues to assess the role of O-GlcNAcylation on AMPAR trafficking during synapse formation and development.43 Interestingly, they found that conditional KO (cKO) of OGT, via transduction with Cre recombinase, decreased the synaptic expression of the GluA2 and GluA3, but not the GluA1 AMPAR subunits.43
Overall, these results clearly suggest a role for O-GlcNAc in the regulation of AMPAR trafficking, perhaps through the direct modification of GluA2, but we have yet to develop a mechanistic understanding of how this might occur. It will be interesting to see whether more sensitive techniques will be able to identify, map, and quantify the dynamics of individual AMPAR subunit O-GlcNAcylation. This information will facilitate the establishment of a casual role of
AMPAR O-GlcNAcylation in regulating trafficking by revealing how O-GlcNAcylation changes at individual sites in response to physiologically relevant stimuli and allowing for the investigation of the effects of expressing glycosylation-deficient AMPAR subunits. Alternatively, it is also possible that O-GlcNAcylation of AMPARs themselves is not responsible for the differences observed in AMPAR trafficking after global changes in O-GlcNAcylation. In fact, several large- scale proteomics studies of O-GlcNAcylated proteins isolated from mouse and human brains revealed multiple PDZ-domain-containing scaffold proteins, SH3 and multiple ankyrin repeat domains (SHANK) 1/2/3, multiple members of the membrane-associated guanylate kinases (MAGUKs), and synaptic Ras GTPase activating protein (SynGAP) 1, are O-GlcNAcylated.30,33-
35,136 PDZ-domain containing scaffolding proteins are known to regulate trafficking and clustering of AMPAR and play a pivotal role in organizing the postsynaptic density and its signaling machinery (also necessary for AMPAR trafficking).137
Thus, perhaps it is not altered AMPAR O-GlcNAcylation itself that is mediating the differences in trafficking outlined above, but rather altered synaptic organization (and hence AMPAR trafficking and clustering132,137) caused by gross perturbations in the O-GlcNAcylation state of many organizational and scaffolding proteins. This might at least partially explain the somewhat conflicting result that decreased O-GlcNAcylation can both increase135 and decrease43 AMPAR insertion into the postsynaptic membrane; if the above hypothesis is correct, small differences in the time course/length of inhibitor treatment, age of the neurons, etc… are likely to lead to large changes in synapse organization (that subsequently effect AMPAR trafficking).132,138 Finally, regardless of whether AMPAR O-GlcNAcylation or changes in the O-GlcNAcylation of scaffolding proteins (certainly not mutually exclusive) are responsible for modulating AMPAR trafficking, it will be important for future studies to systematically and consistently investigate the
role of O-GlcNAcylation (either globally or site-specifically) on regulating AMPAR subunits (both individually and in concert) given the diverse roles of each subunit in different contexts.132,139
Closely related to AMPAR trafficking is LTP and LTD of excitatory synapses. In fact, many of the studies outlined above showed differences in LTP/LTD when O-GlcNAcylation levels were globally altered. As previously mentioned, Tallent et al. found that OGA inhibition increased LTP while the opposite was true with OGT inhibition.42 However, Taylor et al. found that OGA inhibition rather caused severe LTD, and Kanno et al. found that OGT inhibition caused increased LTP, both in acute rat hippocampal slices.40,135 Moreover, in an extensive characterization of OGA hemizygous mice, shown to have increased levels of O-GlcNAcylation in the brain, Suh and colleagues found that both LTP and LTD were attenuated in response to relevant stimuli in acute hippocampus slices.140 Here, the authors suggest that these impairments may be due to the markedly altered phosphorylation dynamics of AMPAR subunit GluA1 at sites critical for LTP and LTD.140 Interpreting these studies in relationship to one another is difficult given the in vivo42,140 versus in vitro40,135 modulation of O-GlcNAc levels and the use of different chemical40,42,135 and genetic140 tools. The result of this is a fairly significant difference in the time between enzyme inhibition and treatment, which is likely to engage differentially engage known compensatory mechanisms.51,81,83 Moreover, the studies almost uniformly used different protocols to alter O-GlcNAcylation, assess LTP and LTD, and investigate mechanisms, further highlighting the need for more systematic studies on the role of O-GlcNAcylation in regulating synaptic plasticity. Last, an alternative explanation for the aforementioned conflicting results might be timescale/treatment dependent, differential reorganization of the global synaptic machinery (resulting from changes in O-GlcNAcylation of key scaffolding/organizational proteins, see above).
Another major aspect of LTP/LTD beyond AMPAR insertion in the postsynaptic membrane is modulation of glutamate release from the presynaptic neuron.141-143 Interestingly, multiple O-GlcNAc sites have been identified on presynaptic proteins involved in regulating presynaptic vesicle trafficking, recycling, and release, e.g. bassoon, piccolo, and synapsin, in multiple large scale O-GlcNAc proteomics experiments.30-35 Both Tallent et al. and Taylor et al.
addressed potential presynaptic mechanisms, although they again reached different conclusions.
Taylor et al. assessed whether glucosamine treatment (demonstrated to significantly increase global O-GlcNAcylation) changed the paired-pulse ratio, an indirect measure of presynaptic vesicle release, in hippocampal slices and found no effect.40 On the other hand, Tallent et al.
described a decrease in paired-pulse facilitation, increased short-term potentiation, and a marked increase in synapsin phosphorylation at multiple sites after OGA inhibition.42 Synapsin, which generally serves to tether presynaptic, neurotransmitter containing vesicles to the neuronal cytoskeleton thereby preventing migration to and fusion with the presynaptic membrane (hence inhibiting neurotransmitter release), is dynamically regulated by phosphorylation in response to neuronal activity.42,144 For example, phosphorylation at serine 9 blocks the association between synapsin and synaptic vesicles while phosphorylation at serine 603 induces a conformational change which decreases synapsin’s affinity for both actin and synaptic vesicles, essentially
“freeing” synaptic vesicles for neurotransmitter release.144 An eloquent followup study by Vosseller and colleagues showed furthermore that O-GlcNAcylation of synapsin I itself, at the single site, Thr87, can directly disrupt the binding between synapsin and synaptic vesicles and decrease the amount of synapsin I localized to synapses.145 Taken together, these studies indicate that O-GlcNAc is playing a key role in regulating synapsin phosphorylation and function to regulate presynaptic release probability and synaptic strength.
Finally, another way that O-GlcNAcylation could be involved in regulating LTP and LTD could be in modulating neuronal membrane potential (and thus controlling neuronal activity). An eloquent study by Yang and coworkers found that the activity of AgRP neurons in the hypothalamus was drastically reduced by OGT KO.75 Two more recent studies by Huganir and colleagues arrived at similar conclusions for α-CaMKII positive neruons in the paraventricular nucleus of the hypothalamus and embryonic excitatory cortical neurons (although the defects here were attributed to impaired development of synaptic spines).43,96 Yang and colleagues hypothesized that the loss of O-GlcNAcylation on potassium voltage-gated channel subfamily KQT member 3 (KCNQ3) may contribute to the decreased neuronal activity observed in OGT KO neurons,75 presumably by changing the baseline excitability and responsiveness to synaptic input of these neurons.146 Interestingly, a subsequent study also showed that increased O-GlcNAcylation also decreases spontaneous activity in excitatory neurons in hippocampal slices.147 In summary, it is clear that changes in global O-GlcNAcylation can have marked effects on neuronal excitability and firing rate, perhaps by regulating the conductance of voltage gated potassium channels (or through other, as of yet unexplored, mechanisms), which would have drastic effects on LTP/LTP on the individual neuron, circuit, and organismal level.146,148,149
Although the evidence for the regulation of molecular and electrophysiological events by O-GlcNAc is being increasingly explored, there are relatively few studies investigating the role of O-GlcNAc in regulating neurobehavioral phenomenon.2,5,40,95,96,140 First, hippocampal overexpression of glycosylation-deficient S40A CREB (versus wild-type CREB) significantly enhanced long-term memory consolidation in a cued auditory fear conditioning paradigm.2 This study represents the potential, critical importance of individual O-GlcNAcylation events in coordinating neuronal signal transduction and gene expression to realize a specific behavior. Other
studies have focused on the behavioral consequences globally altering O-GlcNAcylation in neurons. Two groups studied the effects of OGT KO on memory acquisition and recall, overcoming the embryonic lethality of OGT KO by crossing floxed-OGT mice with mice expressing Cre recombinase, either constitutively5 or inducibly96, under the α-CaMKII promoter (which is not active until postnatal day 14-21). Interestingly, these studies both found very different behavioral phenotypes, hyperphagia96 and increased anxiety and impaired contextual and cued fear leaning (discussed further in the neurodegeneration section below).5 Similarly, Xie et al.
showed that decreasing O-GlcNAcylation pharmacologically also impaired both contextual and cued fear conditioning in mice.95 On the other hand, increased O-GlcNAcylation, due to OGA hemizygosity, also impaired spatial learning and memory in mice as assessed by the Barnes circular maze test and contextual fear conditioning.140 However, increasing O-GlcNAcylation through pharmacological inhibition of OGA did not affect the performance of rats in a contextual fear learning task, although they did demonstrate profound defects in novel object recognition and novel object preference tasks.40
These comparable disruptions in learning and memory observed with decreasing and increasing O-GlcNAcylation suggest that dynamic O-GlcNAc cycling, rather than the presence or absence of the modification, on certain proteins is critical for neuronal function and cognition. Yet, outside of the study of glycosylation-deficient CREB mentioned above,2 almost nothing is known about which specific O-GlcNAcylation events are most important for regulating behavioral phenomena or how they might act to do so. In summary, although we have only begun to explore the role of O-GlcNAc in regulating behavioral phenomena such as memory, appetite, and cognition, the few, exciting studies to date suggest that this is an area ripe for further exploration.