Acute Increases in Protein O-GlcNAcylation Dampen Epileptiform Activity in Hippocampus

Abstract

O-GlcNAcylation is a ubiquitous and dynamic post-translational modification involving the O-linkage of ␤-N-acetylglucosamine to serine/threonine residues of membrane, cytosolic, and nuclear proteins. This modification is similar to phosphorylation and regarded as a key regulator of cell survival and homeostasis. Previous studies have shown that phosphorylation of serine residues on synaptic proteins is a major regulator of synaptic strength and long-term plasticity, suggesting that O-GlcNAcylation of synaptic proteins is likely as important as phosphorylation; however, few studies have investigated its role in synaptic efficacy. We recently demonstrated that acutely increasing O-GlcNAcylation induces a novel form of LTD at CA3-CA1 synapses, O-GlcNAc LTD. Here, using hippocampal slices from young adult male rats and mice, we report that epileptiform activity at CA3-CA1 synapses, generated by GABA A R inhibition, is significantly attenuated when protein O-GlcNAcylation is pharmacologically increased. This dampening effect is lost in slices from GluA2 KO mice, indicating a requirement of GluA2-containing AMPARs, similar to expression of O-GlcNAc LTD. Furthermore, we find that increasing O-GlcNAcylation decreases spontaneous CA3 pyramidal cell activity under basal and hyperexcitable conditions. This dampening effect was also observed on cortical hyperexcitability during in vivo EEG recordings in awake mice where the effects of the proconvulsant pentylenetetrazole are attenuated by acutely increasing O-GlcNAcylation. Collectively, these data demonstrate that the post-translational modification, O-GlcNAcylation, is a novel mechanism by which neuronal and synaptic excitability can be regulated, and suggest the possibility that increasing O-GlcNAcylation could be a novel therapeutic target to treat seizure disorders and epilepsy.

Increased O-GlcNAcylation rapidly decreases GABAAR currents in hippocampus yet depresses neuronal output

“…Surprisingly, we show that O-GlcNAcylation modulates the final output of CA1 pyramidal cells by reducing the action potential probability through intrinsic and synaptic mechanisms. These results position protein O-GlcNAcylation as a potent regulator of both synaptic inhibition as well as excitation, as we reported previously (Stewart et al, 2017; consistent with a decrease in intrinsic excitability.…”

“…As a further test of the effects of increasing O-GlcNAcylation on the net activity in the intact circuit, we performed a complementary experiment measuring the output of the CA1 population by recording extracellular population spikes (pSpike) in the CA1 pyramidal cell layer during a 10 min application of GlcN+TMG (Fig 5Ci, 10-15min). Consistent with the single-cell recordings, we observed a significant and sustained reduction in pSpike amplitude following an increase in O-GlcNAcylation (Fig 5Cii, p = 0.004, paired t-test), which supports our previous report of decreased CA3 output in the context of increased protein O-GlcNAcylation (Stewart et al, 2017).…”

“…Additionally, phosphorylation of to spike coupling is thought to depend on several factors including IPSP magnitude and timing (Pouille and Scanziani, 2001), as well as passive and active properties of the post-synaptic neuron (Magee, 2000). Interestingly, (i) individual pyramidal cells vary widely in their threshold afferent stimulation required for spiking, but this threshold is controlled by the magnitude of the EPSC and not the amplitude or timing of inhibition (Pouille et al, 2009);and (ii) increasing O-GlcNAcylation leads to a depression of field EPSPs in area CA1, likely owing to an internalization of GluA2 containing AMPARs (Hwang and Rhim, 2019;Stewart et al, 2017). Thus, it stands to reason that the decrease in AP probability despite the increase in E/I ratio following increased O-GlcNAcylation could stem from a concomitant depression of the CA3-CA1 EPSC.…”

“…As such, O-GlcNAcylation is positioned to operate as a nutrient sensor (for review, see Lagerlöf, 2018). Additionally, O-GlcNAcylation plays a crucial role in pathological states such as diabetes (Erickson et al, 2013;Vaidyanathan and Wells, 2014) where its elevation can contribute to disease pathogenesis, in epilepsy where increasing O-GlcNAc is protective (Sánchez et al, 2019;Stewart et al, 2017), and in neurodegeneration (Levine etal., 2019;Wang et al, 2016;Yuzwa and Vocadlo, 2014;Yuzwa et al, 2008Yuzwa et al, , 2012Yuzwa et al, , 2014aYuzwa et al, , 2014bZhu et al, 2014), where increasing O-GlcNAc can decrease pathological accumulation of phosphorylated tau or α-synuclein. Collectively, studies published by us and others suggest that maintaining proper balance in O-GlcNAcylation is critical to maintaining normal hippocampal function, as too much or too little O-GlcNAc has been linked to deficits in learning and memory (Taylor et al 2014;Wang et al 2016).…”

“…Recent work (Hwang and Rhim, 2019) showing that increased O-GlcNAc suppresses excitatory transmission at CA3-CA1 synapses through the removal of GluA2 containing AM-PARs is consistent with our findings. Additionally, increased O-GlcNAcylation dampens picrotoxin-induced epileptiform activity in area CA1 and CA3, and reduces seizure activity in the pentylenetetrazole in vivo model of seizure activity in mice (Stewart et al, 2017). Deletion of OGT in αCaMKII expressing neurons in the adult rodent brain causes a reduction in excitatory synaptic input onto hypothalamic PVN neurons (Lagerlöf et al, 2016), while OGT knock-out (KO) specifically from hypothalamic AgRP neurons reduces excitability via its effect on voltage-gated potassium channels (Ruan et al, 2014).…”