O-GlcNAc transferase regulates excitatory synapse maturity Olof Lagerlöfa,b, Gerald W. Hartb, and Richard L. Huganira,1 aSolomon H. Snyder Department of Neuroscience and Kavli Neuroscience Discovery Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; and bDepartment of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Contributed by Richard L. Huganir, December 30, 2016 (sent for review November 10, 2016; reviewed by Vann Bennett and Peter Penzes) Experience-driven synaptic plasticity is believed to underlie adaptive ion channels composed of different combinations of subunits behavior by rearranging the way neuronal circuits process informa- GluA1–4 and conduct the majority of the fast excitatory neuro- tion. We have previously discovered that O-GlcNAc transferase (OGT), transmission in the brain (26). Spines have been reported to in- an enzyme that modifies protein function by attaching β–N-acetylglu- corporate AMPA receptors early in their development (13, 27, 28). cosamine (GlcNAc) to serine and threonine residues of intracellular Apart from additional roles in long-lasting synapses, AMPA re- proteins (O-GlcNAc), regulates food intake by modulating excitatory ceptor activity has been associated with the stabilization of imma- synaptic function in neurons in the hypothalamus. However, how ture spines, possibly, at least in part, via actin-dependent pathways OGT regulates excitatory synapse function is largely unknown. Here (4, 14, 29–35). we demonstrate that OGT is enriched in the postsynaptic density of In neurons, the O-GlcNAc pathway has emerged recently as excitatory synapses. In the postsynaptic density, O-GlcNAcylation on critical for coupling cellular function to energy availability through multiple proteins increased upon neuronal stimulation. Knockout of nutrient-dependent flux via the hexosamine biosynthesis pathway the OGT gene decreased the synaptic expression of the AMPA recep- (HBP), of which the OGT donor substrate uridine diphosphate tor GluA2 and GluA3 subunits, but not the GluA1 subunit. The num- (UDP)-GlcNAc is the end product (36–40). Unlike complex gly- ber of opposed excitatory presynaptic terminals was sharply reduced cans present on the outside of cells and in the secretory pathway, upon postsynaptic knockout of OGT. There were also fewer and less O-GlcNAc is a highly dynamic sugar that is added and removed mature dendritic spines on OGT knockout neurons. These data iden- repeatedly over the lifespan of a single peptide chain. It is tify OGT as a molecular mechanism that regulates synapse maturity. expressed mainly on the inside of cells in the nucleus, cytoplasm, and mitochondria. Only two enzymes regulate its cycling; O-GlcNAc | OGT | excitatory synapses | AMPA receptors O-GlcNAc transferase (OGT) attaches O-GlcNAc to proteins covalently, whereas O-GlcNAcase (OGA) hydrolyses O-GlcNAc euronal synapses, the cell–cell junctions over which neurons from proteins. The brain is one of the organs where O-GlcNAc is Ncommunicate, are formed and eliminated throughout life, the most abundant (39, 41–43). In the synapse, a myriad of and their turnover has for decades been associated with the way proteins carry O-GlcNAc (44–46). Many of these are critical for neuronal circuits adapt to environmental challenges to optimize synaptic plasticity, like alpha calcium/calmodulin-dependent ki- behavior (1, 2). In both humans and animals, early development is nase II (αCaMKII) and SynGAP (46, 47). O-GlcNAc signaling characterized by massive generation of new synapses. About half of does affect learning and memory (48, 49). Acute inhibition of all synapses are then lost during adolescence (2, 3). Most mature OGT or OGA pharmacologically, indicated that O-GlcNAc reg- excitatory synapses occur on dendritic protrusions called spines ulates LTP and LTD by affecting AMPA receptor trafficking. – and essentially all spines contain an excitatory synapse (4 6). In However, there have been contradictory reports of whether global vivo imaging of individual spines for days to months has shown that elevation or depression of O-GlcNAc levels have a stimulatory adult spines are largely stable but a small subpopulation remains or inhibitory effect, respectively, on excitatory synaptic function, plastic (3, 7) and spine turnover is increased by novel experience possibly due to nonspecific effects of OGT inhibitors or differ- – (5, 8 10). Whereas most new spines are thin and withdraw rapidly, ences in mode and length of application of drugs against OGA some enlarge and form stable synaptic contacts (2, 3, 7, 11–14). In fact, the stabilization of a subset of new spines correlates with Significance behavioral performance in several different tasks in multiple ani- mal species (13, 15–17). Rather than synapse formation or density, the selection of which spines are retained once formed has been Neurons in the brain adapt to environmental challenges by suggested to match circuit architecture with behavior (18). Without rearranging their connections, or synapses, to other neurons. affecting spine formation, deleting β-adducin, which regulates ac- This process is called synaptic plasticity and it is unclear how synaptic plasticity is regulated. Here we show that the enzyme tin, perturbed the process by which nascent spines establish func- O-GlcNAc transferase (OGT) modulates the maturity of excit- tional synapses and impaired long-term memory (19). Fragile X atory synapses. The function of OGT depends on the metabolic syndrome, a common form of mental retardation, exhibits a higher status of the cell and the body. Previously, OGT has been linked than normal density of spines but more of them exhibit an im- to appetitive behavior, learning and memory, and neurode- mature morphology and their turnover is not affected by sensory generative diseases like Alzheimer’s disease. Our data suggest experience (9, 20). Conversely, the protein Telencephalin arrests that OGT may underlie brain function by coordinating experi- the maturation of spines and its removal enhances several forms of ence-driven synaptic plasticity with nutrient availability. learning (13, 21, 22). Although many molecules have been identified that affect syn- Author contributions: O.L., G.W.H., and R.L.H. designed research; O.L. performed research; apse number, it is unclear how newly formed spines mature into O.L., G.W.H., and R.L.H. analyzed data; and O.L., G.W.H., and R.L.H. wrote the paper. synapses (13, 23, 24). On a cellular level, learning and memory are Reviewers: V.B., Duke University Medical Center; and P.P., Northwestern University. associated with long-term potentiation (LTP) and long-term de- Conflict of interest statement: G.W.H. receives a share of royalties received by Johns pression (LTD) of neurotransmission. It is widely believed that Hopkins University (JHU) on sales of the CTD 110.6 antibody, which are managed by JHU. LTP and LTD involve synaptic insertion and removal, respectively, 1To whom correspondence should be addressed. Email: [email protected]. α of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. receptors (25). AMPA receptors are glutamate-gated tetrameric 1073/pnas.1621367114/-/DCSupplemental. 1684–1689 | PNAS | February 14, 2017 | vol. 114 | no. 7 www.pnas.org/cgi/doi/10.1073/pnas.1621367114 Downloaded by guest on September 25, 2021 (49–51). We developed a mouse model where OGT was deleted A B genetically from mature brain neurons in vivo and demonstrated H P1 S1 P2 S2 SPM PSD H P1 S1 P2 S2 SPM PSD MW 4 that OGT regulates normal food intake, at least in part, by mod- 225 ncOGT * ulating excitatory synaptic function in neurons in the hypothalamus 3 (36). Using electrophysiology, we observed that knocking out OGT 102 2 sharply reduced the frequency of miniature excitatory postsynaptic 52 currents (mEPSCs). mEPSC amplitude was also decreased, but to a 1 (norm. to hom.) lesser degree. These findings indicate that OGT can underlie 38 OGT expression 0 adaptive behavior partly by regulating normal excitatory synaptic O-GlcNAc Hom. PSD function (36). OGT PSD-95 PSD D pH pH Here we investigate how OGT regulates excitatory synaptic Synaptophysin 3 10 310 function by taking advantage of primary neuronal cell culture MW where OGT is deleted genetically either broadly or sparsely in OGT neurons. We discovered that OGT deletion selectively reduced OGA the synaptic expression of the GluA2 and GluA3 subunits, two O-GlcNAc PSD-95 major AMPA receptor subunits. Mimicking our electrophysio- C Control Bicuculline logical finding of lower mEPSC frequency in vivo, removal of 100 OGT in vitro led to fewer mature morphological synapses and 80 fewer dendritic spines. The spines that were present on OGT KO 60 neurons were largely immature. Collectively, our observations 40 suggest that OGT is important for the maturation of excitatory puncta (%) 20 OGT+ PSD-95 synapses, at least in part through modulating GluA2/3 expression. 0 D The regulation of synapse maturity by OGT represents a model OGT OGT / PSD-95 / MAP2 OGT / PSD-95 / MAP2 for how neuronal circuits may respond to environmental chal- Fig. 1. OGT is enriched in the postsynaptic density of excitatory synapses. lenges, such as nutrient fluctuations, to accommodate behavior. (A) Wb of PSD fractions from brain. The Right blot shows the same sample run again and overexposed. (B) Quantification of OGT expression in the PSD Results relative to the whole-cell